EMBO/SourceData huggingface dataset:
Figure 5(A-B) Western Blot analysis of pCREB protein levels in Atg5flox:CAG-CreTmx WT/ KO cultured neurons at DIV14, treated with forskolin (WTDMSO set to 100%, WTForskolin: 214.17± 48.87%, KODMSO: 63.26± 15.85%, KOForskolin: 33.50± 10.84%, pWT DMSO/WT Forskolin=0.042, pWT Forskolin/KO Forskolin=0.002, two-way ANOVA with Tukey's multiple comparisons). n=4. The same lysates were run in parallel to detect pCREB and CREB and normalized to their respective loading control before calculating the pCREB/CREB ratio.(I, J) Representative fluorescent images and subsequent analysis of primary cortico-hippocampal neurons containing nuclear pCREB either under DMSO-treated condition or under condition when amino acids and serum starvation was induced for 16 hours in the absence or presence of 1µM of PKA inhibitor H89. Neurons were identified by their polarized morphology in DIC. White rectangular boxes indicate panel magnified to the right. In the inserts, LUT gradient was added to highlight the pCREB intensity in the nucleus. Scale bar: 50 µm. DMSO: 52.50±2.757, starvation: 70.00±2.697, starvation+H89: 53.92±3.147; pDMSO/starvation<0.0001, pstarvation/starvation&H89=0.0004, one-way ANOVA with Tukey's multiple comparisons test, n=30 images from n=3 independent experiments.
Figure 6(A-B) Western Blot analysis of PKA substrates carrying RRXS/T motif in primary cortico-hippocampal Atg5flox:CAG-CreTmx WT/KO neurons at DIV14, treated with Forskolin (WTDMSO set to 100%, WTForskolin: 291.85± 17.02%, KODMSO: 152.97± 25.26%, KOForskolin: 182.56± 31.89%, pWTDMSO/WTForskolin: 0.001, pWTForskolin/KOForskolin: 0.033, two-way ANOVA with Tukey's multiple comparisons).(K-M) Representative STED images and Pearson's colocalization coefficient analysis of SHANK3 (K) and CASKIN1 (L) and PSD95 in Atg5flox:CamKIIα-Cre:Ai9 WT/KO neurons. Scale bar: 5µm, 1µm (small panels). WTSHANK3: 0.324±0.022, KOSHANK3: 0.415±0.024; p=0.0067, two-tailed unpaired t-test. nWT=46, nKO=46. WTCASKIN1: 0.255±0.024, KO CASKIN1: 0.326±0.025; p=0.0432, two-tailed unpaired t-test. nWT=39, nKO=43 from n=2 independent experiments.
Figure 7(C,D) Analysis of GLUR1 fluorescent levels at the spines of 10-11 week-old Atg5wt/wt:Ai9 and Atg5flox/flox:Ai9 mice injected with CamKIIα-Cre AAV into the hippocampus. Scale bar: 5µm. GLUR1 level at PSD95-positve spines were assessed measuring mean gray values after background subtraction (WT: 64.55±7.988%, KO: 99.14±6.863%; p=0.0029, two-tailed unpaired t-test). nWT=13, nKO=14 images from n=3 mice (>31 dendritic spines analyzed per each mouse). sp, spine.(G) Representative fluorescence images of Atg5flox:CAG-CreTmx WT/KO neurons transfected with eGFP or eGFP-ATG5 and immunostained for GLUR1, PSD95 and GFP. (H) The overexpression of eGFP-ATG5 in Atg5flox:CAG-CreTmx KO neurons diminished the of GLUR1/PSD95 colocalization compared to the eGFP-overexpressing KO neurons (KOeGFP: 70.07±1.473%, KOeGFP-ATG5: 65.42±1.721%,; p=0.048, two-tailed unpaired t-test). nKO+GFP=42, nKO+GFP-ATG5=50 images from n=5. (I, J) The coexpression of eGFP-PKA R1α and eGFP-PKA R1β in cultured Atg5flox:CAG-CreTmx WT neurons results in significantly increased colocalization between GLUR1 and PSD95 compared to the eGFP-expressing neurons (WT: 42.30±2.453%, KO: 51.32±2.624%, p=0.014, two-tailed unpaired t-test). nWT+GFP=46, nWTGFP+PKA R1 OE=46 images from n=3. Analysis in H, J is performed using a unbiased custom-written Plugin for ImageJ.(K-M) GCAMP7f responses to tetanic stimulation (four tetani, 100 APs at 100Hz, 3s interval) in cell bodies of CamKIIα-Cre-transduced primary cortico-hippocampal Atg5wt:wt/Ai9 (WT) and Atg5flox:flox/Ai9 (KO) neurons expressing mCamKIIα-jGCaMP7f and treated either with DMSO (K) or with 10 µM CNQX for 5 min (L). Autophagy deficient cells showed significantly increased facilitation of GCAMP7f signal to electrical stimulation (ΔF/Fpeak1) compared to the WT, a phenotype which was blocked by the application of CNQX (WTDMSO: 0.28± 0.12, WTCNQX: -0.07± 0.03, KODMSO: 3.74± 0.86, KOCNQX: -0.014± 0.13, p WT DMSO/ KO DMSO<0.0001, pKO DMSO/KO CNQX<0.0001, two-way ANOVA with Tukey's multiple comparisons test). nWT DMSO= 25, nWT CNQX= 27, nKO DMSO= 33, nKO CNQX= 30 neurons from n=3.
Figure 1Schematic overview of the sample collection procedure for single-cell PCR (scPCR). Single IHC cytoplasms from acutely dissected organs of Corti of C57BL/6J (Wt) mice after hearing onset were aspirated and processed for scPCR as depicted. (B') Expression analysis of endophilins-A1-3 from individually isolated IHC cytoplasms using RT-PCR from a representative experimental run. Please note that for these experiments, negative bath control samples from before and after the isolation procedure were an essential requirement to ensure lack of contamination from cellular debris in bath solution. HPRT was used as a housekeeping gene.Immunoblotting of tissue lysates from p15 Wt (C57BL/6J), 1-SKO, 1/2-DKO and 1/3-DKO revealed protein expression of endophilins-A1 and -A2 in the murine cochlea and ensured antibody specificity. Unfortunately, none of the commercially available endophilin-A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts, but rather appeared to (also) detect A1. γ-adaptin was used as loading control. All antibody epitopes localize to the distinct C-terminal regions of the different endophilin-A family members. Hi, hippocampus; Ce, cerebellum; Co, pooled cochleae from a single individual of the indicated genotype; Md, modioli (micro-dissected and pooled from 10 Wt animals); OC, organs of Corti (micro-dissected and pooled from 10 Wt animals).
Figure 2Ca2+-current-voltage relationships evoked by incremental 15 ms step depolarizations from -87 mV to +58 mV revealed a ~25% reduction of ICa in 1/3 DKO and TKO mice. (A') Due to the perinatal lethality of TKO mice, detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically-cultured organs of Corti after 7 days in vitro (DIV). C57BL/6J (Wt) and 1/3-DKO served as controls. Please note that all recordings from cultured IHCs were performed at [Ca2+]e of 10 mM to maximize IHC exocytic performance. (A") Quantification and statistical analysis of individual maximum ICa amplitudes (Imax) of the respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).Ca2+-current inactivation was probed by test pulses of 100 ms to the Imax potential and revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt and 1/3-DKO cells. (B') Quantification and statistical analysis of the residual current at the end of the test pulse. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).Representative ICa (upper panel) and Cm (lower panel) in response to a 50 ms depolarizing step to the potential eliciting Imax. (C') Exocytic ∆Cm and corresponding QCa elicited by depolarizations of stimulus durations from 2-100 ms for all respective genotypes and at [Ca2+]e = 6 mM for a second set of recordings from wild-type IHCs to experimentally approximate the decreased ICa observed in the endophilin mutants. (C") Magnification of the initial, short depolarizing steps (2-20 ms) for clarity. Exocytic ∆Cm of cultured endophilin-deficient IHCs appears strongly reduced. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Tables S2 and S3 for detailed statistical analysis). The reduced Ca2+-efficiency of exocytosis (∆Cm/QCa) in endophilin-deficient IHCs indicates that diminished Ca2+-influx cannot fully account for the reduction of exocytosis. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Table S4 for detailed statistical analysis).
Figure 3Ca2+-current-voltage relationships in response to 10 ms step-depolarizations. (A') The peak of Ca2+-influx was significantly reduced in endophilin 1-SKO (**p = 0.0024) and 1/3-DKO (***p < 0.0001) when compared to Wt. (Wt n = 30/ N = 20; 1-SKO n = 15/ N = 9; 1/3-DKO n = 39/ N = 20; Non-parametric K-W with post-hoc Dunn's correction).Quantification of CtBP2-labeled ribbons revealed a number of approximately 14-15 ribbon synapses per IHC in all groups, that remained comparable across all genotypes (Wt n = 12/ N = 9; 1-SKO n = 6/ N = 4; 1/3-DKO n = 8/ N = 6; one-way ANOVA, p = 0.717).Quantification of the cross-sectional area of CaV1.3 immunofluorescence revealed approx. 34 % smaller clusters in 1-SKOs and 24 % smaller clusters in 1/3-DKOs. (Wt n = 153/ N = 2; 1-SKO n = 102/ N = 2; 1/3-DKO n = 441/ N = 4; ***p < 0.0001; K-W and Dunn's post-hoc test).Representative maximum projections of confocal sections from organs of Corti of p15 Wt, 1-SKO, and 1/3-DKO mice stained for CtBP2 to label synaptic ribbons. Please note that CtBP2 expression is also found in the nucleus (nuclei are highlighted by dashed circles in the individual panels). Scale bars: 10 µm.2D STED images of IHC ribbon synapses stained for CtBP2 (magenta) and CaV1.3 (green). CaV1.3 Ca2+-channels remain clustered at AZs of 1-SKO and 1/3-DKO IHCs. Scale bars: 1 µm.
Figure 4Representative Ca2+-currents (upper panel) and exocytic membrane capacitance increments (∆Cm; lower panel) in response to a 200 ms depolarizing step to -14 mV.Exocytic ∆Cm (top) and corresponding Ca2+-current integrals QCa (bottom) of Wt and endophilin-deficient IHCs in response to voltage steps from -84 to -14 mV of variable stimulus duration (5 to 200 ms). In 1/3-DKO IHCs, our data indicate reduced sustained exocytosis to a strong depolarization (200 ms; *p = 0.0470, K-W with Dunn's post-hoc test; please also refer to Appendix Tables S5 and S6 for detailed statistical analysis). Inset: Initial ∆Cm showed comparable RRP exocytosis in endophilin-deficient IHCs.Ca2+-efficiency to drive exocytosis was reduced for strong depolarizations (200 ms) in 1/3-DKO IHCs; *p = 0.0323, K-W with Dunn's post-hoc test; please also refer to Appendix Table S7 for detailed statistical analysis.
Figure 5Confocal maximum projections of otoferlin-immunolabeled organs of Corti from p15 Wt and 1/3-DKO mice illustrated with an intensity-coded look-up table where brighter colors indicate higher intensity. Scale bars: 5 µm.Otoferlin levels were reduced by approx. 28.4 ± 3.5 % in 1/3-DKO IHCs compared to Wt IHCs (relative intensities normalized to Wt; Wt n = 31 images (229 IHCs)/ N = 5 organs of Corti (3 animals); 1/3-DKO n = 20 images (196 IHCs)/ N = 5 organs of Corti (4 animals); unpaired student's t-test; ***p < 0.001; box plots illustrate the median with the interquartile range, whiskers indicate 10-90 % of data points, the squares present the respective mean value).Normalized otoferlin intensity line profiles through single IHCs of the respective genotypes at a longitudinal central plane through the nucleus from apical (0.0) to basal (1.0) revealed similar distribution patterns of otoferlin in IHCs of Wt and 1/3-DKO and an overall reduction of otoferlin levels throughout the entire cell in 1/3-DKOs. Traces indicate the mean pixel intensity ± SEM; Wt n = 123/ N = 3; 1/3-DKO n = 124/ N = 3.Interaction of otoferlin and endophilin-A1 detected by co-IP in HeLa cells co-expressing GFP-otoferlin and RFP-endophilin-A1. Otoferlin-GFP was immunoprecipitated (IP) by GFP-Trap beads, and blots were probed with a KO-validated anti-endophilin-A1 antibody.Exogenously overexpressed GFP-otoferlin was immunoprecipitated using GFP-Trap beads and incubated with purified endophilin-A1 (pA1). IP was then followed by immunoblotting with an anti-endophilin-A1 antibody.
Figure 6Endocytosis was assessed by determining the decrease in Cm during 20 s post-depolarization in perforated patch recordings from both Wt, 1/2-DKO and 1/3-DKO IHCs. The two kinetic components are presented as average ∆Cm recordings after (A) 20 ms (Wt n = 12/ N = 8; 1/2-DKO n = 13/ N = 7; 1/3-DKO n = 13/ N = 10) and (B) 200 ms (Wt n = 12/ N = 8; 1/2-DKO n = 13/ N = 6; 1/3-DKO n = 14/ N = 10) depolarization stimuli.Average time of return to baseline obtained by fitting a linear function to the Cm-data following a 20 ms depolarization.Average slope of the linear component of endocytosis for 20 ms and 200 ms depolarizing pulses, obtained from fitting Cm-data for 20 s after cessation of the 20 ms depolarizations or for the last 10 s of the Cm- recording for 200 ms depolarizations. (*p < 0.05; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's).Average amplitude of the exponential component obtained from fits to Cm-data following 200 ms depolarization (nexp fit = 11 for Wt, 10 for 1/2-DKO and 11 for 1/3-DKO; x data points in (E) correspond to IHCs with no exponential component in Cm). correspond to statistically identified outliers.time constant of the exponential component obtained from fits to Cm-data following 200 ms depolarization
Figure 7Representative electron micrograph illustrating the region of interest for analysis within a radius of r = 1 µm from the ribbon center (exemplary for a 1/3-DKO specimen). Graphical aids indicate small vesicles, ELVs and different subpopulations of coated structures quantified in (B-D). Scale bar: 500 nm.While an increased number of cytosolic vesicles can be observed at 1-SKO AZs, reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs. RA-SVs were excluded from this quantification.Accumulations of ELVs occur in 1/2-DKO and 1/3-DKO IHCs, as measured by the relative cumulative ELV area per section.Increased overall number of coated structures in endophilin mutants. (D') Prominent accumulation of coated pits can be observed in 1/2-DKO and 1/3-DKO IHCs alongside a shift towards more coated vacuoles (d > 70 nm) in 1/2-DKO IHCs and 1/3-DKO IHCs.
Figure 8Representative electron micrographs of random ultrathin sections from IHC ribbon synapses of p15 mutants lacking different endophilin-A alleles. Scale bars: 200 nm.Reduced numbers of RA-SVs in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001. Reduced ratio of RA-SVs in the distal half of the ribbon over RA-SVs in its proximal half in 1/2-DKO and 1/3-DKO IHCs. K-W followed by Dunn's test; **p < 0.01; ***p < 0.001.Reduced numbers of MP-SVs (D) in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001.Significantly reduced numbers of RA-SVs in 1/2-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0229).Significantly reduced numbers of MP-SVs in 1/2-DKO and a strong trend (p = 0.0511) towards less MP-SVs in 1/3-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0138).Increased SV diameter in 1/2-DKO, but unchanged SV diameter in 1/3-DKOs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0154).Accumulation of ELVs with a minimal distance to the ribbon of less than 200 nm in 1/2-DKOs (one-way ANOVA followed by Tukey's post-hoc test; ***p = 0.0002).
Figure 9Representative electron micrographs of Wt and 1/2-DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity (radius r = 1 µm from the center of the ribbon) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K+. Scale bars: 200 nm.Significantly reduced numbers of RA-SVs in IHCs of 1/2-DKO compared to Wt after stimulation, as quantified using random ultrathin sections of ribbon-occupied AZs from several IHCs per genotype. Mann-Whitney test; ***p < 0.001.Numbers of MP-SVs were slightly reduced in stimulated Wt compared to stimulated 1/2-DKOs, where SV numbers closely matched the ones of unstimulated 1/2-DKO. Mann-Whitney test; *p < 0.05.Reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs compared to Wt after stimulation. RA-SVs were excluded from this quantification. Student's t-test; ***p < 0.001.Stimulated 1/2-DKOs exhibited accumulations of ELVs in comparison to stimulated Wt. Mann-Whitney test; ***p < 0.001.Increased overall number of coated structures in stimulated endophilin 1/2-DKOs. (F') Both, coated vesicles and coated pits forming at ELVs were significantly increased in 1/2-DKO after stimulation. Mann-Whitney test; **p < 0.01; ***p < 0.001.
Figure 1(A) CHO cells were grown in a control medium (see Materials and methods) or starved for 3 or 13 h, after which they were incubated in 50 μM DHE and visualized as detailed in Materials and methods.(B) CHO cells were grown as in (A), treated with 5 nM MitoTracker Red for 30 min at 37°C, washed, treated with 30 μM DCFDA and visualized as detailed in Materials and methods.(C) HeLa cells grown in 96‐well plates were deprived of serum (αMEM), completely starved (EBSS) or maintained in a control medium (α-MEM, 10% FCS) for 2 h, after which they were treated with DCFDA as in (B) and subsequently analyzed in a fluorimeter, as explained in Materials and methods.(D) Data collected from the fluorometric measurements were analyzed as detailed in Materials and methods.
Figure 2(A) Left panel: HeLa cells were starved for 2 h in the presence or absence of either 100 nM wortmannin or 10 mM 3MA, after which they were treated with DCFDA and analyzed by a fluorimeter as described above. Right panel: the rate of degradation of long‐lived proteins was measured in cells incubated in either α‐MEM medium or EBSS medium in the absence or presence of 100 nM wortmannin or 10 mM 3MA. Values are represented as the means±s.d. of three separate determinations.(B) WT MEFs or Atg5 (−/−) MEFs from two separate clones were starved for 2 h before treatment with DCFDA and visualization or fluorometric analysis.
Figure 3(A) Upper panel: CHO cells stably transfected with GFP‐GATE‐16 were preincubated in the presence or absence of 10 mM NAC or 1000 μ/ml catalase for 10 min before starvation for 2 h in the presence or absence of these drugs, or grown in a control medium containing the drugs for 2 h. The cells were then fixed, permeabilized and stained with anti‐GFP monoclonal antibodies. Representative images are shown. Lower panel: HEK 293 cells were transfected with GFP‐GATE‐16. At 24 h post‐transfection, the cells were treated with NAC or catalase and starved as explained above, lysed in Ripa buffer and 100 ‐g of each lysate was separated on 10% SDS-PAGE and subsequently analyzed with anti‐GFP antibodies to detect the transfected GATE‐16 and anti‐tubulin antibodies as control. The data were quantified using NIH image program and are depicted as the percentage of lipidated protein from the total GATE‐16. (*) indicates non‐lipidated and (**) indicates lipidated GFP‐GATE‐16.(B) CHO cells treated with NAC or catalase and starved as detailed in (A) were incubated with DCFDA and visualized by a confocal microscope or analyzed by a fluorimeter as in Figure 1.(C) The rate of degradation of long‐lived proteins was measured in CHO cells incubated in either α‐MEM medium or EBSS medium, or following pretreatment with 10 mM NAC for 10 min or with 1000 μ/ml catalase overnight. A representative experiment is shown.
Figure 4(A) CHO cells (for mammalian Atg8s) or cells of S. cerevisiae (for ScAtg8) were incubated in starvation medium (EBSS or SD‐N, respectively) for different time periods as indicated before isolation of RNA by the Tri‐reagent as explained in Materials and methods. For each gene, data obtained from non‐starved cells were set to an arbitrary value of 1, and results from the corresponding starved cells were normalized accordingly. Results represent the means±s.d. of three separate experiments.(B) HEK 293 cells were grown in control medium or starved for 2 h in EBSS in the absence or presence of 100 nM bafilomycin A1. Lysates (100 μg) obtained in Ripa buffer were run on 12% SDS-PAGE and analyzed by Western blot, using anti‐GATE‐16 or anti‐tubulin antibodies.(C) CHO cells were grown in a control medium in the presence or absence of 1 mM H2O2 for 1 h, or starved for 3 or 13 h. Lysates (10 μg) obtained in Ripa buffer were incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer (25 mM Tris, pH 7.4, 50 mM KCl) at 30°C for 45 min, in the presence or absence of 1 mM DTT. The reaction was stopped by addition of sample buffer and boiling, after which the samples were resolved on 15% SDS-PAGE and subsequently analyzed by Western blot, using anti‐His monoclonal antibodies. The experiment was repeated six times; a representative blot is shown.(D) Left panel: HeLa cells transfected with Myc‐GATE‐16‐HA or with an empty vector as control were labeled with [35S]methionine for 10 min and lysed immediately or chased for 1 h before lysis in Ripa buffer. Lysates were immunoprecipitated using anti‐Myc antibodies and the immunoprecipitates were resolved on 15% SDS-PAGE. Middle panel: HeLa cells transfected with Myc‐GATE‐16‐HA were kept in control medium or starved for 30 min or 13 h in EBSS before labeling with [35S]methionine for 10 min, immediate lysis, analysis by SDS-PAGE and quantification (right panel) using NIH image program. Values are presented as the average percentage of unprimed form out of the total GATE‐16. (*) in all sections of this figure indicates non‐cleaved His6‐GATE‐16‐HA or Myc‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16 or Myc‐GATE‐16.
Figure 5(A) Cleavage activity was tested by incubation of recombinant His6‐HsAtg4A (0.1 μg) and His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence of indicated concentrations of DTT followed by Western blot analysis, using anti‐His monoclonal antibodies. The resulting bands from three separate experiments were quantified with a densitometer using the Bio‐Rad Multi‐Analyst program and are presented as the average percentage of cleaved form out of the total GATE‐16. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.(B) His6‐HsAtg4A was incubated in the presence of 200 μM DTT at 4°C for 10 min. Reduced His6‐HsAtg4A (0.1 μg) was then incubated in 50 KT (to obtain 15 μM DTT) with the indicated concentrations of H2O2 at 25°C for 5 min, after which recombinant His6‐GATE‐16‐HA (0.3 μg) was added and incubation proceeded at 30°C for 45 min. Reaction mixtures from three separate experiments were analyzed and are presented as explained in (A).(C) His6‐HsAtg4A (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) after the following procedures: no treatment (lane 1); pretreatment with 200 μM DTT at 25°C for 5 min (lane 2); treatment with 200 μM DTT followed by treatment with 1 mM H2O2 at 25°C for 5 min (lane 3); and treatment with 200 μM DTT followed by treatment with 1 mM H2O2 for 5 min and then 2 mM DTT (lane 4). Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies.
Figure 7(A) Recombinant His6‐HsAtg4AWT, His6‐HsAtg4AC77A or His6‐HsAtg4AC81S (0.1 μg) was incubated with His6‐GATE‐16‐HA (0.3 μg) in 50 KT reaction buffer at 30°C for 45 min in the presence or absence of 1 mM DTT. Reaction mixtures were analyzed by Western blot using anti‐His monoclonal antibodies. (*) indicates non‐cleaved His6‐GATE‐16‐HA and (**) indicates cleaved His6‐GATE‐16.(B) His6‐HsAtg4AC81S (0.1 μg) was incubated with recombinant His6‐GATE‐16‐HA (0.3 μg) following the same procedure as detailed in Figure 5C. Reaction mixtures were analyzed and are presented as explained in (A).