Journal: Molecular cell

Article Title: Ubiquitin profiling of lysophagy identifies actin stabilizer CNN2 as a target of VCP/p97 and uncovers a link to HSPB1.

doi: 10.1016/j.molcel.2022.06.012

Figure Lengend Snippet: Figure 2. CNN2 translocates to damaged lysosomes and is ubiquitylated for timely dissociation (A) Dynamic association of endogenous CNN2 with damaged lysosomes in HeLa cells. Immunofluorescence of CNN2 and Gal3 as lysosomal damage marker after mock or LLOMe treatment for indicated time periods. Note CNN2 translocation and dissociation before Gal3 clearance. (B) Graph represents the percentage of CNN2 and Gal3-positive vesicles among all Gal3-positive vesicles per cell. More than 30 cells were quantified per condition in each experiment (n = 4 biologically independent experiments). One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test, ** p = 0.0036, *** p = 0.0002, and **** p < 0.0001. Error bars represent the mean ± SEM. (C) Schematic domain structure of CNN2 with positions of identified ubiquitylation sites indicated. CH domain, calponin homology domain; ABS1/2, actin- binding sites. (D) HeLa cells expressing CNN2-GFP wild type or harboring lysine-to-arginine substitutions in the CH domain (CH-KR) following mock or LLOMe treatment as indicated. Note that CNN2 wild type dissociates from LAMP1 vesicles within 3 h, but the ubiquitylation mutants persist. See Figure S3A for CNN2-GFP 5xKR covering the 5 ubiquitylation sites detected by MS. (E) Quantification of (D). Percentage of LAMP1 vesicles positive for CNN2. More than 20 cells were quantified. One-way ANOVA with Tukey’s multiple comparison test, *** p = 0.0003 and **** p < 0.0001; ns, not significant. Error bars represent the mean ± SD. (F) Live-cell imaging of HeLa cells expressing CNN2-GFP CH-KR and mCherry-Gal3. Lysosomes were loaded with photosensitizer AIPcS2a, irradiated in the indicated area, and chased over the course of 1 h. See Figure S3E for wild type and CNN2 5xKR imaging data. (A, D, and F) Scale bars, 10 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rat monoclonal anti-Gal3 (IF, 1:500) Santa Cruz Biotechnology Cat#sc-23938; RRID:AB_627658 Rabbit monoclonal anti-LAMP1 (IF, 1:500) Cell Signaling Technology Cat#9091; RRID:AB_2687579 Rabbit polyclonal anti-CNN2 (IF, 1:500; WB, 1:1000) Thermo Fischer Scientific Cat#PA5-61878; RRID:AB_2639249 Mouse monoclonal anti-CNN2 (WB, 1:1000) Origene Cat#TA503688; RRID:AB_11124845 Rabbit polyclonal anti-LC3 (IF, 1:500; WB, 1:1000) MBL Cat#PM036; RRID:AB_2274121 Rabbit polyclonal anti-p62 (IF, 1:500; WB, 1:1000) Sigma-Aldrich Cat#P0067; RRID:AB_1841064 Mouse monoclonal anti-p62 (WB, 1:1000) Abnova Cat#A00008878 Mouse monoclonal anti-HSP27 (IF, 1:500) Thermo Fischer Scientific Cat#MA3-015; RRID:AB_325463 Rabbit polyclonal anti-HSP27 (WB, 1:1000) Thermo Fischer Scientific Cat#PA5-78010; RRID:AB_2735924 Rabbit polyclonal anti-TAX1BP1 (WB, 1:1000) Sigma Cat#HPA024432; RRID:AB_1857783 Mouse monoclonal anti-p97 (WB, 1:2000) Santa Cruz Biotechnology Cat#sc-57492; RRID:AB_793927 Mouse monoclonal anti-P4D1 (WB, 1:1000) Cell Signaling Technology Cat#3936; RRID:AB_331292 Mouse monoclonal anti-Tubulin (WB, 1:5000) Sigma-Aldrich Cat#T-5168; RRID:AB_477579 Mouse monoclonal anti-GFP (WB, 1:10000) Roche Cat#11814460001; RRID:AB_390913 horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (WB, 1:10000) Biorad Cat#170-6515; RRID:AB_11125142 horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (WB, 1:10000) Biorad Cat#1706516; RRID:AB_11125547 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 568 (IF, 1:500) Invitrogen Cat#A11011; RRID:AB_143157 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 488 (IF, 1:500) Life technologies Cat#A11034; RRID:AB_2576217 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#A21071; RRID:AB_2535732 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#10696113 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 594 (IF, 1:500) Thermo Fisher Scientific Cat#A-11032; RRID:AB_2534091 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#10246252 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#A-11006; RRID:AB_2534074 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 568 (IF, 1:500) Thermo Fisher Scientific Cat#A-11077; RRID:AB_2534121 (Continued on next page) e1 Molecular Cell 82, 2633–2649.e1–e7, July 21, 2022

Techniques: Marker, Translocation Assay, Comparison, Binding Assay, Expressing, Live Cell Imaging, Irradiation, Imaging

Journal: Molecular cell

Article Title: Ubiquitin profiling of lysophagy identifies actin stabilizer CNN2 as a target of VCP/p97 and uncovers a link to HSPB1.

doi: 10.1016/j.molcel.2022.06.012

Figure Lengend Snippet: Figure 7. p97 and HSPB1 cooperate in removing ubiquitylated CNN2 from lysosomes (A) HSPB1 is trapped on CNN2 in the absence of p97 activity. Proximity biotinylation analyzed by western blot in indicated conditions of lysosome damage (LLOMe) and p97 inhibition (NMS-873). Cells expressing CNN2-APEX2 were treated with LLOMe for 1 h and chased for 2 h after washout prior to the addition of H2O2 to trigger biotinylation. (B) Loss of p97 or HSPB1 function impairs CNN2 dissociation in a non-additive manner. The time course of CNN2 localization to damaged lysosomes upon p97 inhibition (NMS-873), HSPB1 depletion, or a combination of both as indicated. Scale bars, 10 mm. (C) Quantification of (B). The graph represents the percentage of CNN2 and Gal3-positive vesicles among all Gal3-positive vesicles per cell. More than 30 cells were quantified per condition in each experiment (n = 3 biologically independent experiments). Two-way ANOVA with Tukey’s multiple comparison test, **** p < 0.0001, *** p = 0.0004, * p = 0.0118. Error bars represent the mean ± SEM. (D) HSPB1 acts downstream of ubiquitylation together with p97. HeLa cells stably expressing CNN2-GFP were LLOMe treated for 1 h and chased for 2 h prior to denaturing lysis upon indicated treatments after p97 inhibition (NMS-873), HSPB1 siRNA, or a combination of both as indicated. Ubiquitylation of CNN2 was assessed by western blot after immunoprecipitation using GFP nanobodies. Note that loss of HSPB1, or p97 inhibition, leads to the increased accumulation of ubiquitylated CNN2 after LLOMe-induced damage, but that effects are not additive. (E) Model. After lysosome damage, various resident proteins become ubiquitylated to serve as an anchor point for autophagy-receptor-mediated recruitment of the LC3-decorated phagophore. CNN2 is recruited by associating with the p62 autophagy receptor and then stabilizes actin filaments that assist phagophore formation. CNN2 needs to be subsequently ubiquitylated and removed by p97 with the help of HSPB1 to allow efficient phagophore formation.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rat monoclonal anti-Gal3 (IF, 1:500) Santa Cruz Biotechnology Cat#sc-23938; RRID:AB_627658 Rabbit monoclonal anti-LAMP1 (IF, 1:500) Cell Signaling Technology Cat#9091; RRID:AB_2687579 Rabbit polyclonal anti-CNN2 (IF, 1:500; WB, 1:1000) Thermo Fischer Scientific Cat#PA5-61878; RRID:AB_2639249 Mouse monoclonal anti-CNN2 (WB, 1:1000) Origene Cat#TA503688; RRID:AB_11124845 Rabbit polyclonal anti-LC3 (IF, 1:500; WB, 1:1000) MBL Cat#PM036; RRID:AB_2274121 Rabbit polyclonal anti-p62 (IF, 1:500; WB, 1:1000) Sigma-Aldrich Cat#P0067; RRID:AB_1841064 Mouse monoclonal anti-p62 (WB, 1:1000) Abnova Cat#A00008878 Mouse monoclonal anti-HSP27 (IF, 1:500) Thermo Fischer Scientific Cat#MA3-015; RRID:AB_325463 Rabbit polyclonal anti-HSP27 (WB, 1:1000) Thermo Fischer Scientific Cat#PA5-78010; RRID:AB_2735924 Rabbit polyclonal anti-TAX1BP1 (WB, 1:1000) Sigma Cat#HPA024432; RRID:AB_1857783 Mouse monoclonal anti-p97 (WB, 1:2000) Santa Cruz Biotechnology Cat#sc-57492; RRID:AB_793927 Mouse monoclonal anti-P4D1 (WB, 1:1000) Cell Signaling Technology Cat#3936; RRID:AB_331292 Mouse monoclonal anti-Tubulin (WB, 1:5000) Sigma-Aldrich Cat#T-5168; RRID:AB_477579 Mouse monoclonal anti-GFP (WB, 1:10000) Roche Cat#11814460001; RRID:AB_390913 horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (WB, 1:10000) Biorad Cat#170-6515; RRID:AB_11125142 horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (WB, 1:10000) Biorad Cat#1706516; RRID:AB_11125547 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 568 (IF, 1:500) Invitrogen Cat#A11011; RRID:AB_143157 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 488 (IF, 1:500) Life technologies Cat#A11034; RRID:AB_2576217 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#A21071; RRID:AB_2535732 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#10696113 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 594 (IF, 1:500) Thermo Fisher Scientific Cat#A-11032; RRID:AB_2534091 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#10246252 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#A-11006; RRID:AB_2534074 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 568 (IF, 1:500) Thermo Fisher Scientific Cat#A-11077; RRID:AB_2534121 (Continued on next page) e1 Molecular Cell 82, 2633–2649.e1–e7, July 21, 2022

Techniques: Activity Assay, Western Blot, Inhibition, Expressing, Comparison, Stable Transfection, Lysis, Immunoprecipitation

Journal: The EMBO Journal

Article Title: The ATG8 E3-like ligases sense lysosomal damage and initiate ESCRT-mediated membrane repair

doi: 10.1038/s44318-025-00672-1

Figure Lengend Snippet: ( A ) Confocal images of HeLa WT, ATG16L1 KO, TECPR1 KO and E3-DKO cells treated with 1 mM LLOMe for 30 min. Nuclei were stained with DAPI. Scale bars = 10 µm. ( B , C ) Quantification of ALIX and Gal3 area ( B ) and colocalization ( C ) from ( A ). Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n ≥ 50 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. ( B ) ns (not significant) represents P > 0.05, *** P = 0.0001. ( C ) ns (WT vs 16KO) P = 0.9997, ns (WT vs TECKO) P = 0.9873, ** P = 0.0030. ( D ) Confocal images of HeLa WT and E3-DKO cells treated with 1 mM LLOMe for 30 min. Scale bars = 5 µm. ( E ) Quantification of IST1 lysosomal recruitment in HeLa WT and E3-DKO cells co-transfected with IST1-EGFP and LAMP1-mCherry and treated with 1 mM LLOMe. Images were acquired every 15 s. Data are presented as mean ± SD from five independent experiments ( n > 25 cells). ( F ) Confocal images of HeLa TECPR1/ATG16L1 DKO cells transfected with the indicated TECPR1 mutant and treated with 1 mM LLOMe for 30 min. Scale bars = 5 µm. ( G ) Western blot analysis of HeLa TECPR1/ATG16L1 DKO cells stably expressing the indicated TECPR1 mutant and treated +/− LLOMe. ( H ) Confocal images of cell lines from ( E ) treated with 1 mM LLOMe for 30 min. Scale bars = 10 µm for whole cell images and 2 µm for insets. ( I ) Quantification of ALIX area from ( H ). Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n = 60 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. ns (− vs ∆1-377) P = 0.7197 , ns (− vs ∆AIR) P = 0.9439, **** P < 0.0001. .

Article Snippet: Gal3 (87985, IF 1:400), ATG12 (2010, WB 1:1000), LC3B (#2775, WB: 1:1000), GABARAPL1 (#26632, WB: 1:1000), GABARAPL2 (14256, WB: 1:1000) and ATG4B (5299, WB 1:1000) were purchased from Cell Signaling.

Techniques: Staining, Transfection, Mutagenesis, Western Blot, Stable Transfection, Expressing

Journal: The EMBO Journal

Article Title: The ATG8 E3-like ligases sense lysosomal damage and initiate ESCRT-mediated membrane repair

doi: 10.1038/s44318-025-00672-1

Figure Lengend Snippet: ( A ) Western blot analysis of HeLa ATG16L1 KO and three ATG16L1/TECPR1 DKO clones treated with or without 1 mM LLOMe for 30 min. ( B ) Confocal images of cell lines from ( A ) treated with 1 mM LLOMe for 15 min and immunostained for ALIX and Gal3. Nuclei were stained with DAPI. Scale bars = 10 µm for whole image and 2 µm for insets. ( C ) Top: Confocal images of HEK WT, ATG16L1 KO, TECPR1 KO and ATG16L1/TECPR1 DKO cells treated with 1 mM LLOMe for 15 min and immunostained for ALIX. Scale bars = 10 µm. Bottom: Quantification of ALIX area. Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n = 60 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. ns (not significant) represents P > 0.05, * P = 0.0314, **** P < 0.0001. ( D ) Western blot analysis of HeLa and HEK KO cell lines treated with or without 1 mM LLOMe for 30 min. .

Article Snippet: Gal3 (87985, IF 1:400), ATG12 (2010, WB 1:1000), LC3B (#2775, WB: 1:1000), GABARAPL1 (#26632, WB: 1:1000), GABARAPL2 (14256, WB: 1:1000) and ATG4B (5299, WB 1:1000) were purchased from Cell Signaling.

Techniques: Western Blot, Clone Assay, Staining

Journal: The EMBO Journal

Article Title: The ATG8 E3-like ligases sense lysosomal damage and initiate ESCRT-mediated membrane repair

doi: 10.1038/s44318-025-00672-1

Figure Lengend Snippet: ( A ) Confocal images of ATG KO cell lines treated with or without 1 mM LLOMe for 30 min and immunostained for ALIX and Gal3. Scale bars = 10 µm for whole image and 5 µm for insets. ( B ) Quantification of ALIX and Gal3 area from ( A ). Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n = 50 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. ns (5KO) P > 0.9999, ns (7KO) P > 0.9999, **** P = < 0.0001. ( C ) Western blot analysis of ATG5 KO cells stably expressing WT HA-ATG5 or HA-ATG5 K130R . ( D ) Confocal images of cell lines from ( C ) treated with 1 mM LLOMe for 30 min and immunostained for ALIX and Gal3. Scale bars = 10 µm. Quantification of ALIX area is shown to the right. Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n = 60 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. ns not significant ( P = 0.9930), **** P < 0.0001. Comparisons against WT are shown. .

Article Snippet: Gal3 (87985, IF 1:400), ATG12 (2010, WB 1:1000), LC3B (#2775, WB: 1:1000), GABARAPL1 (#26632, WB: 1:1000), GABARAPL2 (14256, WB: 1:1000) and ATG4B (5299, WB 1:1000) were purchased from Cell Signaling.

Techniques: Western Blot, Stable Transfection, Expressing

Journal: The EMBO Journal

Article Title: The ATG8 E3-like ligases sense lysosomal damage and initiate ESCRT-mediated membrane repair

doi: 10.1038/s44318-025-00672-1

Figure Lengend Snippet: ( A ) Schematic outline of lysosome rupture assay. Scale bars = 10 µm. ( B ) Quantification of ALIX and Gal3 area from ( A ). Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n = 60 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. Comparisons are shown against the cell line-matched untreated sample. ns (not significant) represents P > 0.05 (WT-LLOMe-Gal3, P > 0.9999; WT-Wash-Gal3, P = 0.3449; E3DKO-LLOMe-Gal3, P = 0.9935; E3DKO-Wash-ALIX, P = 0.9962; 5KO-LLOMe-Gal3, P = 0.9976; 5KO-Wash-ALIX, P = 0.9919; 8KO-LLOMe-Gal3, P > 0.9999; 8KO-Wash-Gal3, P > 0.9999), * P = 0.0191, ** P = 0.0028, *** P = 0.0001, **** P < 0.0001. ( C ) Schematic outline of lysosome repair/re-acidification assay. Scale bars = 10 µm. ( D ) Quantification of LysoTracker Red puncta from ( C ). Grey points represent individual cells from three independent experiments. Colored points represent the means of individual experiments ( n = 60 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. ns (not significant) represents P > 0.05 (WT 45 min, P = 0.0654; E3DKO 45 min, P = 0.9955; E3DKO 90 min, P = 0.3624; 5KO 45 min, P = 0.9825; 5KO 90 min, P = 0.1703; 8KO 45 min, P = 0.9996; 8KO 90 min, P = 0.8363), **** P < 0.0001. .

Article Snippet: Gal3 (87985, IF 1:400), ATG12 (2010, WB 1:1000), LC3B (#2775, WB: 1:1000), GABARAPL1 (#26632, WB: 1:1000), GABARAPL2 (14256, WB: 1:1000) and ATG4B (5299, WB 1:1000) were purchased from Cell Signaling.

Techniques:

Journal: The EMBO Journal

Article Title: The ATG8 E3-like ligases sense lysosomal damage and initiate ESCRT-mediated membrane repair

doi: 10.1038/s44318-025-00672-1

Figure Lengend Snippet: ( A ) Western blot analysis of HeLa WT and ALG2 KO cells treated with 0.5 mM LLOMe for 15 min. ( B ) Representative confocal images of HeLa WT and ALG2 KO cells treated with or without 0.5 mM LLOMe for 15 min and immunostained for IST1 and LAMP1. Scale bars = 10 µm. ( C ) Quantification of IST1 cell area from ( B ). Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n ≥ 45 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. ns (not significant) ( P = 0.1393), *** P = 0.0002, **** P < 0.0001. ( D ) Quantification of IST1/LAMP1 colocalization from ( B ). Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n = 45 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a Student’s t test. ns (not significant) represents P > 0.05. ( E ) Representative confocal images of HeLa WT and ALG2 KO cells treated with or without 0.25 mM LLOMe for 5 min and immunostained for Gal3. Scale bars = 10 µm. ( F ) Quantification of Gal3 puncta from ( B ). Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n ≥ 72 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. ns (not significant) (UNT, P > 0.9999; +LLOMe, P = 0.8105), **** P < 0.0001. .

Article Snippet: Gal3 (87985, IF 1:400), ATG12 (2010, WB 1:1000), LC3B (#2775, WB: 1:1000), GABARAPL1 (#26632, WB: 1:1000), GABARAPL2 (14256, WB: 1:1000) and ATG4B (5299, WB 1:1000) were purchased from Cell Signaling.

Techniques: Western Blot

Journal: The EMBO Journal

Article Title: The ATG8 E3-like ligases sense lysosomal damage and initiate ESCRT-mediated membrane repair

doi: 10.1038/s44318-025-00672-1

Figure Lengend Snippet: ( A ) Confocal images of HeLa WT, ATG5 KO, E3-DKO and ATG8 KO cells treated with or without BAPTA-AM followed by 1 mM LLOMe for 30 min and immunostained for CHMP2A and Gal3. Scale bars = 10 µm. ( B ) Quantification of CHMP2A/Gal3 cell area from ( A ). Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n = 60 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. ns (not significant) represents P > 0.05, * P = 0.0134. ( C ) Confocal images of HeLa WT, ATG16L1 KO and TECPR1 KO cells treated with or without BAPTA-AM followed by 1 mM LLOMe for 30 min and immunostained for CHMP2A. Scale bars = 20 µm. ( D ) Quantification of CHMP2A cell area from ( C ). Small points represent individual cells from three independent experiments. Large points represent the means of individual experiments ( n = 60 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one-way ANOVA with Tukey’s multiple comparisons tests. ns (not significant) represents P > 0.05, *** P = 0.0009. .

Article Snippet: Gal3 (87985, IF 1:400), ATG12 (2010, WB 1:1000), LC3B (#2775, WB: 1:1000), GABARAPL1 (#26632, WB: 1:1000), GABARAPL2 (14256, WB: 1:1000) and ATG4B (5299, WB 1:1000) were purchased from Cell Signaling.

Techniques: