EMBO/SourceData huggingface dataset:
Figure 1(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8 were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A),(A, B) Wild-type (WT) (BY4741) cells expressing GFP−Atg8were grown to mid-log-phase in YPD and were then preincubated with 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (-) in the rich medium for 30 min. Cells were washed and shifted to nitrogen-starvation medium (SD-N) for 4 h in the presence of 50 µM cerulenin or 50 µM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies (A), or were visualized by fluorescence microscopy (B)(C) Cells (TOS038) expressing Atg1-GFP were grown to mid-log phase and were then preincubated in YPD with 50 µM cerulenin or with DMSO for 30 min. Cells were washed, shifted to SD-N for 4 h, and then visualized by fluorescence microscopy. Scale bar, 5 μm.(D) WT (BY4741) and pep4∆ (TOS015) strains were grown to mid-log phase and preincubated in YPD with cerulenin or DMSO for 30 min. Cells were pulse-labeled for 10 min with [35S] methionine and cysteine and chased for the indicated time periods. Cell lysates were subjected to immunoprecipitation with anti-Ape1 antibodies followed by SDS‒PAGE and X-ray film to detect radioactive signals. prApe1, premature Ape1; mApe1, mature Ape1. ((E) Cells of the fas1∆ (TOS029) strain expressing GFP‒Atg8 were grown to mid-log phase in YPD + 0.1 mM palmitic/stearic/myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min. Cells were then shifted to SD-N for the indicated times. Cell lysates were subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies.(F) WT (BY4741) cells were grown to mid-log phase and then preincubated in YPD with 25 µM or 50 µM cerulenin (25, 50) or with DMSO (-) in rich medium for 30 min. Cells were washed and shifted to SD-N in the presence of cerulenin (25, 50) or DMSO (-). Cells were lysed and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP and anti-Pgk1 antibodies. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type YPD, complete medium.
Figure 2(A) Cells (TN124 strain) were grown to mid-log phase and preincubated in YPD with 50 µM cerulenin for the indicated time periods (Cer preincubation) or incubated with DMSO without preincubation] (-). Cells were then shifted to SD-N for 3 h with the addition of DMSO or 50 µM cerulenin (cer). Autophagic activity was measured by alkaline phosphatase assay. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).(B) Cells were grown as in (A), stained with BODIPY, and visualized by fluorescence microscopy. Scale bar, 5 μm.(C) WT (SCY62) and tagΔsteΔ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 3 h. Cells were stained with Nile red and visualized by fluorescence microscopy. Scale bar, 5 μm.(D) WT (SCY62) and tag∆ste∆ (H1246) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cell lysates were subjected to SDS−PAGE, followed by western blot analysis using anti-GFP, anti-Ape1 (prApe1, premature Ape1; mApe1, mature Ape1), and anti-Fas1 and anti-Pgk1 antibodies.(E) WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. cer, cerulenin; DIC, Differential interference contrast; FA, fatty acids; SD-N, nitrogen-starvation medium; WT, wild-type, YPD, complete medium.
Figure 3(A) GAL-DGA1GAL-ARE2lro1Δ are1Δ (FYS118 strain) was grown on SC (synthetic minimal medium without dextrose) + raffinoseovernight, diluted to OD 0.4, and grown to mid-log phase in either SC + glucose or SC + galactose medium. Cells were shifted to SD-N for 2 h, stained with BODIPY, and visualized by fluorescencemicroscopy. Scale bar, 5 μm.(B) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 were grown as in A. Cells were lysed at the indicated times and subjected to SDS‒PAGE, followed by western blot analysis using anti-GFP antibodies. ((C, D) WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP−Atg8 (C) or GFP−Atg1 (D) were grown as in A and visualized by fluorescence microscopy after 2 h in SD-N. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild-type.
Figure 4(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 6 h. Cells were stained with BODIPY and visualized by fluorescence microscopy. Scale bar, 5 μm.
Figure 5(A) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. (B) Quantification of the GFP/GFP−Atg8 ratio. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). (. (C) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells expressing GFP-Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. ((D) WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226) and ste∆ (H1112) cells were grown as in (A). Lysates were subjected to SDS−PAGE in urea gel, followed by western blot analysis using anti-Atg8 and anti-Pgk1 antibodies. Atg8 I, non-lipidated Atg8; Atg8 II, lipidated Atg8. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.
Figure 6Fig. 6 Lack of lipid droplets inhibits starvation-induced formation of autophagosomes. (A, H) WT pep4∆ (pep4∆) (A,B), tag∆ste∆ pep4∆ (tag∆ste∆pep4∆)(C,D), ste∆ pep4∆ (ste∆pep4∆)(E,F) and tag∆ pep4∆ (tag∆pep4∆) (G,H) cells were grown to an exponential phase in YPD before being starved in SD-N for 2 h, and were then processed for electron microscopy. H is a magnification of the box in panel G. Magnification of proliferating ER in a tag∆ste∆ (H1246) cell is shown in (I) Asterisks indicate autophagic bodies. N, nucleus; PM, plasma membrane; CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; V, vacuole; #, trehalose; F, ER proliferation. Scale bars: 1 µm (A-H) and 200 nm (I). (J) Average number of autophagic bodies per cell section was determined by counting 100 randomly selected cell profiles. Error bars represent standard deviations fromcounting of the three grids. ***P < 0.001 (Student's t-test). SD-N,nitrogen-starvation medium; WT, wild type YPD, complete medium.
Figure 7(A) WT (BY4741), tgl1∆, yeh1∆ ,and yeh2∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS−PAGE, followed by western blot analysis using anti-GFP antibodies. Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).(B) WT (BY4741) and tgl3∆tgl4∆tgl5∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test). ((C) WT (BY4741), ayr1∆ and ldh1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05 (Student's t-test).(D) WT (BY4741), ldh1∆ayr1∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. **P < 0.01 (Student's-t test).(E) WT (BY4741), ice2∆ and ldb16∆ cells expressing GFP−Atg8 were grown, treated, lysed, and western blotted as in (A). Quantification of the GFP/GFP−Atg8 ratio is presented on the right. Error bars represent the s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 ***P < 0.001 (Student's t-test).(F) WT (BY4741), ice2∆ and yeh1∆ cells expressing GFP−Atg8 were grown to mid-log phase in YPD and shifted to SD-N for 4 h. GFP−Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. DIC, Differential interference contrast; SD-N, nitrogen-starvation medium; WT, wild type.
Figure 1(A) Total lysates of different mouse organs underwent immunoblotting analysis using anti-Pdia4 and anti-actin antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.(C) Min6 cells were treated with glucose and palmitate at the indicated dosages. Total lysate underwent immunoblotting analysis using the indicated antibodies. (D) Human islets were treated with glucose and palmitate at the indicated dosages, followed by immunoblotting analysis. ( Data information: Data from 3 experiments are presented as the mean ± SD.(G) Sera from wild-type (WT) BKS and Leprdb/db mice, aged 4, 8, and 18 weeks, B6 mice, fed with a normal diet (ND) and a high fat diet (HFD) for 4 weeks, and humans, healthy volunteers and diabetic patients (T2D), were quantified for Pdia4 level using an anti-Pdia4 ELISA kit. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.(H) Immunoblotting analysis of Pdia4 and markers in the cytosolic (Cyto), nuclear (Nuc), membrane (Mem), mitochondrial (Mito), and ER compartments of Ming 6 cells using the indicated antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.
Figure 2(A) Survival rate and life span of WT, Pdia4-/-(KO), and Pdia4tg/tg (TG) mice on Leprdb/db background from birth to 120 weeks Data information: The number of mice (n) is indicated in parentheses. Data from over 3 experiments are presented as the mean ± SD. Log rank test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.(B) Pancreata of 18-week-old WT and Pdia4-/- (KO) mice on BKS or Leprdb/db background were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.(D) The same batch mice as in (B) were given water containing BrdU. The BrdU+ cells of the islets were visualized and quantified (BrdU+ labeling). TUNEL-positive cells in the islets of the mice (B) were visualized and quantified (TUNEL assay). BrdU+ cells per islet area (0.01 mm2) and TUNEL+ cells per islet area (0.05 mm2) are expressed in arbitrary units (AU). The dash circles indicate islet regions and the black arrowheads indicate TUNEL+ cells. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.F) insulin in supernatants of the mouse islets (F) in GSIS assays. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3) were used in GSIS assays. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
Figure 3(B) Pancreata of the same batch of mice as in (A), at the age of 14 weeks, were stained with anti-insulin (αIns) antibody and DHE (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar = 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.(D) The islets of the mice from (C) were tested for GSIS. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3 mM) were used in GSIS assays. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
Figure 4(A) The islets of WT, Pdia4-/- (KO) and Pdia4tg/tg (TG) BKS mice were isolated and grown in complete DMEM medium containing 3.3 mM (LG) and 30 mM glucose (HG) for 12 h. The islets stained with propidium iodide (PI), photographed, and quantified. Scale bar = 50 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.The islets from the mice (A) were incubated with MitoGreen plus MitoSOX in the presence of glucose at 3.3 mM (LG) and 16.7 mM (HG). Mitochondrial ROS in the islets were visualized and quantified. Scale bar = 100 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.The islets from the mice (A) were incubated with Hoechst 33342 (Ho) plus CM-H2DCFDA in the presence of glucose at 3.3 mM (LG) and 16.7 mM (HG). cytosolic ROS in the islets were visualized and quantified. Scale bar = 100 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.(E) Mitochondria isolated from the mouse islets (A) were measured for the activity of ETC CI (left), CII/III (middle) and CIV (right) using MitoCheck assays. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
Figure 5(A) Ultrastructure of mitochondria and insulin granules in typical β-cells of 3 mouse lines on BKS and Leprdb/db backgrounds. Arrows indicate the mitochondria whereas arrowheads indicate insulin granules. Scale bar = 1 μm. Data information: A total of 15-23 images per group were analyzed. Data from each group are presented as the mean ± SD.
Figure 6(E) The construct encoding Flag-Pdia4 or its mutants and that expressing Myc/Flag-tagged Ndufs3 (left) or p22phox (right) were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis. N-terminal Flag tag, catalytic domains (a, a' and a''), non-catalytic domains (b and b'), and C-terminal KEEL are indicated. Pdia4* is a mutant of Pdia4 whose 3 CGHC motifs were changed into 3 SGHS motifs (a*, a'* and a''*).(F) The construct encoding Myc/Flag-tagged p22phox, Ndufs3 or their mutants and that expressing Flag-tagged Pdia4 were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis using anti-Flag antibody.
Figure 7(A) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK), a Pdia4 RNAi (KD) and a Pdia4 cDNA (OVE) were sorted and tested for the Pdia4 protein level (left). The cells were incubated with NAC (1 mM) and then stained with MitoSOX or CellROX in response to 0.5 mM (LG) and 25 mM (HG) glucose for an additional 30 min. Signal from MitoSOX (middle) and CellROX (right) was re-plotted into histograms. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.(B) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK) and an RNAi of Ndufs3 (Ndufs3 KD) or p22phox (p22phox KD) were selected and tested for levels of Ndufs3 (1st column), mitochondrial ROS (2nd column, MitoSOX), p22phox (3rd column), and cytosolic ROS (4th column, CellROX). The cells were grown in the presence of 0.5 mM (LG) and 25 mM (HG) glucose. (C) The same experiments as (B) were conducted except that Min6 cells were infected with a lentivirus expressing a cDNA of truncated Ndufs3 (tNdufs3) and p22phox (tp22phox). ( Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
Figure 8(D) 293T cells, which were transfected with the construct encoding Flag-Pdia4 and that expressing Myc/Flag-tagged p22phox or Ndufs3, were treated with GHTT (28 μM) for 30 min. The cells were lysed and incubated with anti-Pdia4 antibodies plus protein G beads. Their total lysates (TL) and immunoprecipitates (IP) underwent immunoblotting analysis with anti-Flag antibody.(F) The sections of pancreata of the mice (E) were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
Figure 1C, Microscale thermophoresis (MST) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711. D, dDATmfc did not show any interactions with NO711 in the MST experiments. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.E, SKF89976a interacts with purified ts5 dDATGAT construct with an affinity of 34 µM. F, MST experiments display no interactions of purified dDATmfc with SKF89976a. The MST profiles shown are one of two independent measurements. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.G, Michaelis-Menten uptake kinetics of 3H-GABA by the GAT1WT used in the study displaying a KM value of 11.4 μM.H, Inhibition of 3H-GABA uptake by the substrate analogue nipecotic acid displaying a Ki value of 14.4 μM. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.I, Inhibition of 3H-GABA uptake through GAT1WT by tiagabine (Ki= 725 nM), NO711 (1.07 μM) and SKF89976a (7.3 μM). Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.
Figure 4B, Substitution of L300 to other hydrophobic side chains and the consequences on transport activity. L300W has the maximal effect on the transport activity. The histogram represents mean of a total 6 measurements (n=6) done as two independent measurements. Individual datapoints are represented as black dots around the error bar that represents s.e.m.Changes to Michaelis-Menten kinetics for L300F mutant that displays weakened 3H-GABA uptake activity with KM = 20.9 μM. A near 10-fold loss of nipecotic acid inhibition potency was observed at a value of 134.3 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows; nipecotic acid,Changes to Michaelis-Menten kinetics for L300F mutant that displays weakened 3H-GABA uptake activity with KM = 20.9 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows panel E-0.0001; tiagabine, panel F-0.0017; NO711, panel G-0.018; SKF89976a
Figure 5E, F, Eadie-Hofstee plots of E, tiagabine and F, NO711 display competitive mode of inhibition of 3H GABA uptake in GAT1WT. G, Eadie-Hofstee plot of SKF89976a displaying mixed (competitive+non-competitive) inhibition of 3H-GABA uptake in GAT1WT. Data information: (E-G) Concentrations of the inhibitors were decided based on the Ki values calculated for individual inhibitors. Each data point was plotted using 6 measurements carried out in two independent measurements in triplicates. Error bars represent s.e.m.
Figure 6C, Michaelis-Menten kinetics of GAT1WT and GAT1 S359E displays a weakened KM value (28.54 ± 2.3 μM). The enhanced Vmax is a consequence of the enhanced expression of the mutant transporter in the assayed cells monitored by FSEC. Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.D, 3H-GABA uptake inhibition by the substrate analogue nipecotic acid for GAT1 S359E displaying a Ki value of 30.1 μM Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.E, The equivalent mutation in dDATWT E384S causes a complete ablation of transport activity despite retaining similar expression levels as dDATWT. Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.
Figure 2HEp-2 cells remained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNF1, CNFY or the N- or C-terminally deleted toxin variants at 37°C for 4 h. The cells were thoroughly washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti-Flag antibody.