In this investigation, the isolate S. halophilum strain LY20 was selected for further study because it appeared to be the best Romidepsin molecular weight producer of extracellular amylase and protease. To date, there are no reports for amylase and protease production at the same time from one isolate, because the protease can hydrolyze other proteins such as amylase. However, maximal production of both enzymes was observed simultaneously during the stationary growth

phase of LY20 (Fig. 2). This particular phenomenon could be explained that the amylase was not the substrate of the protease, which was confirmed by SDS-PAGE after incubating the two enzyme solutions (80 °C and pH 10.0) for 30 min (data not shown). There are many reports on isolation of amylases from halophiles (Mellado et al., 2004; Litchfield, 2011), but pure preparation of halophilic β-amylase has not been obtained. In this study, purification of an β-amylase from LY20 was reported. Similar enzyme was previously described from Halobacillus sp. LY9 (Li

& Yu, 2011), but its enzymatic properties were mostly obtained from crude extracts. Molecular weight of the β-amylase was determined to be 81 kDa (Fig. 3, lane 2). Cyclopamine purchase The value was higher than other β-amylases from nonhalophiles (Shen et al., 1988; Young et al., 2001). The enzyme showed an optimal activity at 70 °C and excellent thermostability under high temperatures. These characteristics made it obviously different from other β-amylases, which were neither

active nor stable at temperatures above 65 °C (Shen et al., 1988; Young et al., 2001). It is desirable that amylases Mannose-binding protein-associated serine protease should be active at high temperature for gelanization (100–110 °C), liquefaction (80–90 °C), and saccharification (60–65 °C) for the application in the starch industry. Until today, amylases from bacteria belonging to genus Bacillus are heavily used in the starch-processing industry (Mamo & Gessesse, 1999; Demirkan et al., 2005). As thermostability is an important feature for amylolytic enzymes, the β-amylase from LY20 might be industrially exploited for starch liquefaction and saccharification. Molecular weight of the purified protease was estimated to be 30 kDa on SDS-PAGE. Similar values presented other halophilic proteases previously characterized (Karbalaei-Heidari et al., 2007a, b; Xiong et al., 2007). The enzyme showed the optimal activity at 80 °C. In contrast to other proteases from halophiles (Amoozegar et al., 2007; Karbalaei-Heidari et al., 2009), it required relatively higher temperature to maintain the maximum activity. Moreover, high thermostability over a wide temperature range (30–80 °C) was observed. These properties made it potential use in industrial applications that require high temperatures. The amylase and protease from LY20 were found to be highly active and stable in the presence of higher concentrations of NaCl.

For example, late presenters may be less likely to adhere to follow-up and/or medication when they do start HAART [11,12], and many of the deaths that occur in late presenters may not be preventable, regardless of HAART initiation, simply because the patient presented for care at too late a stage for treatment to be effective [13]. Furthermore, patients starting HAART rapidly after diagnosis may continue to be investigated for symptoms that were present at diagnosis – the underlying clinical event may often only be diagnosed some time later, after treatment has been initiated. Our aim was to determine whether factors associated with late presentation to care

services influence treatment responses independently of a low CD4 cell count. We therefore compared outcomes of HAART in individuals who Z-VAD-FMK cell line presented and commenced therapy with CD4 cell counts <200 cells/μL with those in individuals who presented with higher CD4 counts but who delayed starting therapy until their CD4 count was <200 cells/μL. We performed a longitudinal analysis of the UK Collaborative HIV Cohort (CHIC) Study, a collaboration of some of the largest HIV clinics selleck inhibitor in the United Kingdom (see Appendix). Participating centres provide routinely collected data on all adult patients

(≥16 years old) attending for care since 1996. The data collected include information on demographics, AIDS events, deaths, antiretroviral

use, CD4 cell counts and HIV RNA levels; the current data set includes information on 32 607 patients seen at 11 clinical centres up to the end of 2007. We identified HIV-infected adults from the UK CHIC database who commenced first-line HAART [defined as a combination that included at least one nucleoside reverse transcriptase inhibitor (NRTI) with either a nonnucleoside Cobimetinib manufacturer reverse transcriptase inhibitor (NNRTI) or a ritonavir-boosted protease inhibitor (PI/r)] from 1 January 1998 to 31 December 2007. Eligible subjects were required to have at least one CD4 cell measurement in the 6 months prior to commencing HAART (where more than one was available, the result closest to HAART initiation was used), a pretreatment viral load (also in the 6 months prior to starting HAART) >500 copies/mL and at least one day of follow-up post-HAART. In order to exclude any bias that may be introduced by the extremely high mortality rate of late presenters in the first few months after diagnosis [14] or the high nonattendance rate of some late presenters, we excluded any individuals who died or who were lost to follow-up within the first 3 months after diagnosis. Our analyses are thus focused on patients who enter a HAART treatment programme which may be reasonably expected to be successful.

From only one bacterial colony, THN1, a potential mlrA gene was amplified and sequenced. blast analysis showed a 98.5% identity between this sequence and the mlrA gene sequence LBH589 of Sphingomonas sp. ACM-3962. The 16S rRNA gene of this bacterial strain was also sequenced, and a homologous search by blastn showed a maximum identity (99%) to Novosphingobium aromaticivorans DSM 12444 (GenBank no. CP000248). Therefore, this bacterial strain was identified as Novosphingobium sp. THN1 belonging to the family Sphingomonadaceae. Removal of microcystin LR in the THN1 culture was observed following analysis of the remaining microcystin LR (Fig. 1). There was a sharp decline during the first 12 h

and 91.2% of the toxin was eliminated in this period. Because microcystin PF-02341066 mw LR could not be detected in the culture after 60 h, complete degradation was concluded.

No decrease in the toxin occurred in the negative control (data not shown). A potential mlr gene cluster with four genes mlrA, mlrB*, mlrC and mlrD was successfully cloned from THN1. All the gene sequences were confirmed to be mlr by aligning with the corresponding genes found in GenBank. The coverage of each mlr sequence from GenBank and their similarity to mlr of THN1 was calculated using bioedit V5.0.6 (Table 2). THN1 had maximum identities with different strains for each gene including mlrA (MD-1, 99.7%), mlrB* (C-1, 96%), mlrC (C-1, 91.7%) and mlrD (ACM-3962, 95.7%). A particularly low similarity (83.7%) of mlrA was found between THN1 and Y2 (Saito et al., 2003), indicating that the Y2 strain has experienced more variation. The two mlr clusters of THN1 and ACM-3962 had a similarity of 95.6%. Relative locations and directions of transcription for each mlr gene of THN1 were the same with ACM-3962. Because the only available mlrC gene sequence (1521 bps) from ACM-3962 does not contain a stop codon, the mlrC (1536 bps) coding 511 amino acid residues, found in this study, was the first reported complete ORF for this gene. Alignment of

mlrB* sequences diglyceride for THN1 and ACM-3962 showed three base insertions (Fig. 2a) at positions 30(C), 44(C) and 1176(G). Apparently, the insert mutations caused a frameshift and eight stop codons (Fig. 2b) within the gene sequence. In an attempt to determine whether mlrB* was transcribed into mRNA in the THN1 cells, we tried to amplify mlrB* from the total cDNA. As displayed in the gel image (Fig. 3), high-quality total RNA was extracted from THN1 cells and no genomic DNA could be detected in the RNA extracts after digesting with DNase. In PCR reactions using total cDNA, the mlrA amplicon was obvious, but no mlrB* product could be detected. In other words, no mRNA of mlrB* gene existed in the complete RNA for the THN1 cells. Upregulated expression of mlrA gene was detected upon exposure to microcystin LR (Fig. 4).

4c). Interestingly, these results showed that increased katG transcription in the rho mutant (Fig. 4b) is not accompanied by an equivalent increase in the levels of KatG protein. These data suggested that low stationary-phase KatG catalase–peroxidase activity in the rho mutant could be due to a deficiency in translation or in post-translational mechanisms such as polypeptide folding or incorporation of the heme cofactor. However, the levels

of immunoreactive KatG in the stationary-phase cells of strain SP3710 are comparable to those in NA1000, indicating that translation of the polypeptide is taking place and suggesting that a reduction in KatG translation efficiency is an unlikely explanation for the drastically decreased KatG activity in the stationary phase. Taken together, our results showed that the rho mutant is under permanent oxidative stress, and exogenous addition of oxidant agents could selleckchem be overwhelming C59 wnt for the cell’s response. We found that KatG activity is severely reduced in the rho mutant, and this seems to be quite a specific effect, because the activities of two SODs were apparently not affected. The decreased activity of KatG could be a result of several contributing effects caused by the rho mutation, either directly via effects on

transcription termination of relevant genes or as an indirect result of the intrinsic oxidative stress status of the cell. The fact that katG transcription is increased in the rho mutant, and catalase– peroxidase protein levels do not differ considerably between the rho mutant and the wild-type strain, suggests that the effect of the rho mutation on KatG is exerted at a translational

or a post-translational level. In the latter case, it remains to be established whether these deficiencies are in improper folding of the protein or defective incorporation of the heme group to make a functional enzyme. We thank Dr Carlos Menck and Raquel Rocha, Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, for assistance with fluorescence microscopy. We thank Mr Eren Sumer, Department of Biochemistry, Albert Einstein College Aspartate of Medicine, for assistance with in situ staining for catalase activity and Dr Regina Baldini for help in the preparation of the anti-KatG antiserum. This work was supported by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) to M.V.M. During the course of this work, V.C.S.I. and V.S.B. were supported by fellowships from FAPESP. M.V.M. is partly supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). “
“The complete DNA sequence of the 41 102-bp plasmid pXap41 from the invasive plant pathogen Xanthomonas arboricola pv. pruni CFBP 5530 was determined and its 44 coding regions were annotated.

Attaining higher coverage rates will require additional influenza vaccination programs in schools, universities, and ethnic medical associations’ PI3K cancer clinics. In addition, wider use of recall and reminder systems can achieve higher coverage among children and adults recommended for influenza vaccination.10 A large percentage of travelers we surveyed became ill either during or within a week after travel (43%), which was similar to the findings of other studies.13–17 The prevalence of ILI among study participants and their companions was close to the prevalence

of diagnosed respiratory infections (7.8%) among returned travelers who visited GeoSentinel network clinics.17 Although the finding was not significant, our study showed that 9 of 11 travelers who developed ILI had not been vaccinated against influenza. A study showed a 25%–34% reduction in ILI prevalence among in-season vaccinated adults.16 Therefore, all travelers should be considered for pre-travel influenza vaccination (both seasonal and H1N1 influenza vaccine) to reduce their risk of infection.10,16 Regarding attitudes toward H5N1 AI, our study found that Asians, FB travelers, those working in occupations other than health care/animal care, and those

who did not seek pre-travel advice were less likely to recognize possible risk factors such as contact with farm animals and birds, and participating in slaughtering and cleaning poultry. Although the risk of H5N1 AI to US travelers is still low,18 clinicians should address avian influenza preventive RAD001 measures, especially among travelers to countries where avian influenza is prevalent in birds and humans. Many travelers are looking for new experiences and adventures, which can increase their risk of exposure to infectious diseases, including novel influenza strains.13–18 We found that many travelers

participated in unplanned activities during their travel, such as visiting rural areas, visiting food markets, and attending large gatherings; thus, clinicians should carefully review travelers’ trip itineraries with the expectation that they might change their plans Histone demethylase and consider the full range of potential activities and risks in the travel destination. Our study corroborated the findings of previous studies regarding the health-seeking behavior of travelers, showing that less than half of travelers reported seeking any type of pre-travel health advice,19–22 and approximately 30% were FB travelers. We found that the primary care practitioner was the most common source of pre-travel health advice among FB travelers, followed by the internet and friends or relatives. In addition, several studies, including our own, addressed the underutilization of travel health specialists for pre-travel health advice, compared with primary health care physicians.

4D, middle panels) than in wild-type neurons (Fig. 4D, upper panels). Addition of HA-Cbln1 to the culture medium restored accumulation of endogenous NRXs associated with GluD2 puncta on cbln1-null Purkinje cell dendrites (Fig. 4D, lower panels). Together, these results indicate that Cbln1/GluD2 serves as a presynaptic Natural Product Library organizer by directly accumulating its presynaptic receptor NRXs(S4+). Cbln1 also serves as a postsynaptic organizer that induces clustering of GluD2 and its

associated proteins at the postsynaptic site. To examine whether NRX functions as a postsynaptic organizer by forming a tripartite complex with Cbln1 and GluD2, we cultured HEK293 cells expressing GluD2 with beads coated with NRX1β. GluD2 clustering was induced around beads coated with NRX1β(S4+) only when HA-Cbln1 was added to the culture medium (Fig. 5A). However, beads coated with NRX1β(S4−) did not cause clustering of GluD2 even in the presence of HA-Cbln1 (Fig. 5A), suggesting that NRX1β(S4+) caused GluD2 clustering in HEK293 cells by forming a complex with Cbln1. The C-terminus of GluD2 interacts directly with several intracellular molecules in neurons; many of these serve as scaffolds for other postsynaptic molecules. Thus, to examine whether NRX also functions Sotrastaurin molecular weight as a postsynaptic organizer in neurons,

we cultured cbln1-null Purkinje cells with beads

coated with NRX1β(S4+) from 10 to 13 DIV. Immunocytochemical analyses showed that GluD2 clustering was induced around beads only in the presence of HA-Cbln1 (Fig. 5B). Similarly, shank2, a scaffold protein that binds to the C-terminus of GluD2, clustered around beads coated with NRX1β(S4+) (Fig. 5B). In contrast, beads coated with NRX1β(S4−) did not cause clustering of GluD2 or shank2 even in the presence of HA-Cbln1 (Fig. 5B). Coimmunostaining of presynaptic synapsin I and postsynaptic GluD2 showed that www.selleck.co.jp/products/Staurosporine.html GluD2 puncta induced by beads coated with NRX1β(S4+) in the presence of HA-Cbln1 were not associated with synapsin I-positive presynaptic terminals (Fig. 5C), indicating that NRX1β(S4+)-beads directly induced GluD2 clustering at the contact sites. These results indicated that the tripartite complex consisting of NRX, Cbln1 and GluD2 serves as a bidirectional synaptic organizer. Of the Cbln family members, Cbln1, Cbln2 and Cbln4 mRNAs are expressed in various brain regions outside the cerebellum, including the olfactory bulb, entorhinal cortex and certain thalamic nuclei (Miura et al., 2006). As NRXs(S4+) are also highly expressed in these regions (Ichtchenko et al., 1995), Cbln family members may also be involved in synapse formation by forming complexes with NRXs.

Therefore, we attempted to construct an in-frame deletion and several gene replacement mutations of nla6S that would not disrupt transcription of genes downstream of nla6S. However, we were unable to construct any of these strains, which is consistent with the idea that Nla6S is important for growth. In summary, our work suggests Nla6S is the founding member of a

new family of HKs found in fruiting members of the Cystobacterineae suborder of the myxobacteria. The goal of future work will be to determine whether Nla6S-like HKs play crucial roles in fruiting body development and to determine whether their mechanisms of action are similar to well-characterized HKs. Zaara Sarwar was OSI-906 manufacturer funded in part by an

International Fellowship from the American Association of University Women (AAUW). This work was supported by National Science Foundation Grant IOS-0950976 to A.G. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“SoxS,MarA, and Rob are homologous transcriptional activators of numerous superoxide- and antibiotic resistance genes but many of the regulated genes are yet to be characterized. In this study, microarrays and RT-PCR analysis were used to show the overexpression of the ompN porin and its upstream gene, ydbK, in an Escherichia coli multidrug-resistant mutant and in a strain constitutive for SoxS. However, transcriptional 17-AAG fusions revealed that SoxS 3-oxoacyl-(acyl-carrier-protein) reductase (not MarA or Rob) only activated the ydbK promoter but not the ompN upstream region. RT-PCR experiments showed the overexpression of a combined ydbK–ompN transcript in the SoxS-overexpressing strain. Surprisingly, a bioinformatic approach revealed no soxbox upstream of the ydbK

promoter. Thus, the ydbK and ompN genes are coexpressed in an operon and are likely activated by SoxS indirectly. It is known that YdbK is involved in superoxide resistance. Thus, individual ompN and ydbK mutants were tested for superoxide susceptibility. Nonetheless, only the ydbK mutant was susceptible to paraquat, a superoxide generator. These mutants, as well as an OmpN-overproducing strain, were further tested for antibiotic resistance. No significant decreased susceptibility was observed. Thus, ydbK plays a role in superoxide resistance but no role for either gene is found in resistance to the antibiotics tested. MarA, SoxS, and Rob of Escherichia coli are highly homologous members of the AraC/XylS family of positive regulators. Overproduction of MarA and SoxS and post-translational activation of Rob are needed to exert their regulatory role (Gallegos et al., 1997). MarA transcription is controlled by the repressor function of MarR (encoded within the marRAB operon; Cohen et al.

In one series, all four tones were drawn from the same harmonic whereas in the other they alternated between an inharmonic and harmonic complex. The harmonic tone complex had a fundamental frequency of 100 Hz, whereas the inharmonic complex had the same fundamental frequency but with each component shifted by the same amount (Δf). A harmonic complex with a 100-Hz fundamental frequency was used as it consists of components

with frequencies around 1000 Hz, where frequency discrimination was measured in Experiment 1. Both complexes had the same harmonic envelope equal to a fundamental frequency of 100 Hz but with different TFS. In each trial, one interval, selected at random, contained the harmonic complex CDK inhibitor and the other contained the inharmonic complex. Intervals were indicated by numbered BGB324 molecular weight flashing boxes presented onscreen coincident with the presentation of the complexes. Subjects clicked with a computer mouse on the box corresponding to the interval containing the inharmonic tones. Following Moore & Sęk (2009), the duration of each complex was 200 ms and the two complexes were separated by a 300-ms interval. Feedback was given after each trial, with the selected observation period flashing either

green for correct or red for incorrect. At the start of each block, Δf was set at 50 Hz and was adapted according to response. Again following Moore & Sęk (2009), blocks were terminated following eight reversals, and the threshold for the block was taken as the arithmetic mean of Δf for the last six reversals. To prevent subjects discriminating on place coding, all components were passed through a fixed band-pass filter set centered at 900 Hz with a width of 110 Hz rolling on at 30 dB per octave. Threshold equalizing noise, presented 15 dB below stimulus presentation level (SPL) and extending from 50 to 11 050 Hz, was used to mask components of the complexes falling outside the band-pass filter.

Following Moore & Sęk (2009), SPL for the harmonic complex was set 20 dB SPL above each subject’s 70.7% absolute threshold measured using an adaptive 2I-2AFC staircase method for 900 Hz immediately prior to each session. The sampled point of the psychometric function of absolute threshold was changed from Experiment 2A for consistency with previously established measures of TFS. To give consistent performance, subjects almost had one initial training session prior to testing where they practised the TFS task for ~45 min. There were two counterbalanced TFS testing sessions after training where either anodal or sham tDCS was applied, separated by a week to avoid any carry-over effects of stimulation. Subjects completed seven threshold procedures during the 20 min of either tDCS or sham stimulation. Each staircase lasted ~2 min, varying with the subject’s response times and number of trials needed for six reversals. The threshold for that session was taken as the arithmetic mean of the seven thresholds for the session, each of which lasted approximately 35 min.

“
“The objective was to examine whether a common polymorphism in the dopamine D4 receptor gene (DRD4) might be a potential biomarker for behavioral variation within the autism spectrum disorder clinical phenotype. Children (N = 66) were evaluated with a validated mother- and

teacher-completed DSM-IV-referenced rating scale. Partial eta-squared (ηp2) was used to gauge the magnitude of group differences: 0.01−0.06 = small, Volasertib solubility dmso 0.06−0.14 = moderate and > 0.14 = large. Children who were 7-repeat allele carriers had more severe oppositional defiant disorder behaviors according to mothers’ (ηp2 = 0.10) and teachers’ (ηp2 = 0.06) ratings than noncarriers, but the latter was marginally significant (P = 0.07). Children who were 7-repeat allele carriers also obtained more severe maternal ratings of tics (ηp2 = 0.07) and obsessions–compulsions (ηp2 = 0.08).

Findings for maternal ratings of separation anxiety were marginally significant (P = 0.08, ηp2 = 0.05). Analyses of combined DRD4 and dopamine transporter gene (DAT1) genotypes approached significance (P = 0.05) for teachers’ ratings of oppositional behavior and mothers’ ratings of tics. DRD4 allelic variation may be a prognostic biomarker for challenging behaviors in children with autism spectrum disorder, but these exploratory findings remain tentative pending replication with larger independent samples. “
“Nontuberculous mycobacteria (NTM) are ubiquitous organisms found in soil, water, and biofilms.

Epigenetics inhibitor Engineered surface topography has been proposed as a method to reduce microbial biofilm formation. The Sharklet® micropattern silicone surface has been shown to reduce biofilm formation of pyogenic bacteria. We hypothesized that this micropattern surface will also reduce colonization Lepirudin by Mycobacterium abscessus, a human pathogen. Smooth and micropattern silicone samples were incubated with 1 × 106 M. abscessus mL−1 for 2 and 4 days. After processing to optimize recovery of adhered mycobacteria, there was a 75% and 50% reduction in the number of viable M. abscessus recovered from the micropattern surfaces compared to the smooth surfaces at 2 and 4 days after inoculation, respectively. Ziehl–Neelsen staining after measures to remove the adherent microorganisms revealed fewer residual M. abscessus on the micropattern samples as compared to smooth samples, validating the quantitative culture results. Microscopic observation of 2, 4, and 8 day M. abscessus cultures on micropattern samples showed that the organisms preferentially colonized within the channels between the rectangular features. In summary, a micropattern surface reduces the colonization of a pathogenic NTM. It remains to be seen whether this micropattern can reduce infections in humans.

These two sequences were flanked by SbfI and SfiI restriction sites, and separated in between by two nonidentical FauI restriction

sites. The three roGFPs were amplified by PCR, adding the respective FauI sites. These constructs were then ligated between the KAR2 leader and the HDEL sequences, and introduced into the same pPuzzle vector as that used for the cytosolic expression. The integration locus for the ER constructs was the 5′ region of the P. pastoris enolase gene. The plasmid containing the gene PDI1 (encoding protein disulfide isomerase; Inan et al., 2006) was generated by PCR using P. pastoris genomic DNA as a template and SbfI and SfiI as restriction sites. The gene was cloned into a pPuzzle vector containing the Zeocin resistance marker, and was expressed under the control of the GAP1 promoter. The vector was integrated into the native PDI1 gene locus AZD6244 of the P. pastoris genome after linearization in the respective sequence. Electrocompetent P. pastoris host strains were transformed using a BioRad Minipulser. Conditions for the pulsing included a cuvette with a 2-mm gap, a charging voltage of 2000 V and a pulse length of 4 ms. After 2-h regeneration on YPD (per liter: 20 g yeast extract, 10 g soy peptone, 20 g glucose), cells were cultivated for 48 h and at 30 °C on YPD-agar this website plates (per liter: 20 g yeast extract, 10 g soy peptone, 20 g glucose, 20 g agar-agar) containing 25 μg mL−1

Zeocin or 100 μg mL−1 Hygromycin (both Invivogen), respectively. Shake-flask experiments were carried out in 100-mL shake flasks incubating at 28 °C at 170 r.p.m. For each strain, 12–15 individual clones were used to inoculate 10 mL of freshly prepared minimal medium. The medium used in these experiments was M2 minimal medium containing per liter: 20 g of glucose, 20 g of citric acid, 3.15 g of (NH4)2HPO4, 0.03 g of CaCl2·2H2O, 0.8 g of KCl, 0.5 g of MgSO4·7H2O,

2 mL of biotin (0.2 g L−1) and 1.5 mL of trace salts stock solution. The pH was set to 5.0 with 5 M KOH solution. Trace salts stock solution contained per liter: 6.0 g of CuSO4·5H2O, Erastin research buy 0.08 g of NaI, 3.0 g of MnSO4·H2O, 0.2 g of Na2MoO4·2H2O, 0.02 g of H3BO3, 0.5 g of CoCl2, 20.0 g of ZnCl2, 5.0 g of FeSO4·7H2O and 5.0 mL of H2SO4 (95–98% w/w). A protocol for the determination of the redox state using rxYFP in S. cerevisiae (Ostergaard et al., 2004) served as a template for the establishment of a redox-measuring procedure in living P. pastoris cells. The culture (840 μL) with an OD of approximately 30 was used for determination of the redox ratio. Redox measurements in the cytosol were performed with and without addition of the cell-solubilizing agent digitonin. Comparison of both experiments yielded the same results; therefore, further experiments were performed without digitonin. For the ER, digitonin was not added to the cells, because it would lead to a whole-cell lysis, which was not desirable in this case.