EMBO/soda-nel huggingface dataset:
NLRC3 expression in dendritic cells attenuates CD4+ T cell response and autoimmunity (2019)
Figure 3

Figure 3. NLRC3 deficiency in DC promoted EAE development.

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE.

A. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).

B. Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide.

C. Recall response to MOG(35-55) by splenocytes isolated from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.

D. Expression of Il12, Il6, Il23 and Il27 mRNA in DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.

E-F. CFSE proliferation assay (E) and cytokine secretion (F) of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.

Data information: Data (n=5 in C, D and F) shown are the mean ±SD. *P < 0.05, **P < 0.01and ***P < 0.001 by an unpaired t-test. Data are representative of three independent experiments with similar results.
NLRC3 expression in dendritic cells attenuates CD4+ T cell response and autoimmunity (2019)
Figure 4

Figure 4 NLRC3 negatively regulated p38 signaling pathway in DC.

DCs were sorted from spleens of WT or Nlrc3-/- mice.

A. Purified DCs were treated with LPS (100 ng/ml) for specified time. DC lysates were probed for phosphorylated p65 (p-p65), total p65, p-AKT, AKT, p-p38, p38, p-ERK, ERK, p-JNK, JNK and GAPDH.

B-D. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). (B) Concentrations of IL-12, IL-6, IL-23 and IL-27 in supernatants were detected by ELISA. Cytokines in culture supernatants (C) and CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.

Data information: Data (n=5 in B and C) shown are the mean ±SD. *P < 0.05, **P < 0.01and ***P < 0.001 by an unpaired t-test. Data are representative of three independent experiments with similar results.
NLRC3 expression in dendritic cells attenuates CD4+ T cell response and autoimmunity (2019)
Figure 5

Figure 5. NLRC3 deficiency in DC promoted EAE development via p38 signaling pathway.

A. Activity phosphorylation of p38 were detected in DCs in the spleens from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction. Pooled data are presented in the right panel.

B-D. DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. (B) Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5). (C) Frequencies of CD4+ T cells that express IFN-γ and IL-17A in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel. (D) Expression of Il12, Il6 and Il23 mRNA in DCs sorted from spleens of DC(WT), DC(NLRC3-KO), DC(p38-KO) and DC(p38+NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.

Data information: Data (n=5 in A, B, C right and D) shown are the mean ±SD. **P < 0.01 and ***P < 0.001 by one-way ANOVA test. Data are representative of three independent experiments with similar results.
NLRC3 expression in dendritic cells attenuates CD4+ T cell response and autoimmunity (2019)
Figure 6

Figure 6. NLRC3-overexpression attenuated antigen-presenting function of DCs.

DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time.

A. DC lysates were probed for p-p38, p38, NLRC3 and GAPDH. Densitometry quantification of band intensity were presented in the right panel.

B. Enzyme-linked immunosorbent assay of cytokines in culture supernatants of DCs treated with LPS for 48 hours.

C-D. Cytokines in culture supernatants (C), and CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.

Data information: Data (n=3 in A right; n=5 in B and C) shown are the mean ±SD. *P < 0.05, **P < 0.01 and ***P < 0.001 by an unpaired t-test. Data are representative of three independent experiments with similar results.
NLRC3 expression in dendritic cells attenuates CD4+ T cell response and autoimmunity (2019)
Figure 7

Figure 7. Vaccination with NLRC3-overexpression DCs suppressed EAE.

EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction.

A. Mean clinical scores of EAE (n= 5 mice per group ). Arrows indicate DC vaccine administration.

B. Effects of therapeutic DC vaccination on B6 EAE. The maximum score: Mean of the maximum scores per mouse in each group. Data shown are the mean ±SD. Determination of statistical differences was performed using by an one-way ANOVA test.

C-D. Recall proliferative (C) and cytokine response (D) to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.

Data information: Data shown (n=5 in A, C and D) are the mean ±SD. **P < 0.01 and ***P < 0.001 by an one-way ANOVA test. Data are representative of three independent experiments with similar results.
Developmental arsenic exposure impairs cognition, directly targets DNMT3A and reduces DNA methylation (2022)
Figure 2

Figure 2 - Arsenic directly binds to DNMT3A.

A LC-ESI-MS/MS spectrum of Dnmt3a pulled down by Biotin-As in NIH3T3 cells. NIH3T3 cells were treated with 10 μg/mL Biotin-As for 2 hours, followed by pull-down assay and determination using LC-ESI-MS/MS.

B-D Validation of the binding between DNMT3A and Biotin-As at 5 μg/mL in NIH3T3 (B), MEFs (C) and CCRF-CEM (D) cells in the Biotin-As pull-down assay.

E Schematic diagram of the structure of human DNMT3A. Upper panel: All cysteines in DNMT3A are shown. Lower panel: Structure of DNMT3A-ADD (PDB: 3A1A) and the four interested cysteine triads.

F Measurement of DNMT3A-ADD thermodynamic stability upon ATO treatment. Purified DNMT3A-ADD was mixed with ATO at the indicated ratios in 20 mM HEPES (pH 7.5) for 1 hour. Melting curves were measured by DSF (left panel), and the calculated ΔTm values are shown (right panel). Data are presented as mean ± SD of n = 4 technical replicates. ****p < 0.0001 by unpaired two-tailed Student's t test.

G LC-MS spectra of purified DNMT3A-ADD before and after ATO incubation at 1:5 molar ratio for 16 hours.

H, I Biotin-As pull-down assays for the transfected DNMTs (H) and DNMT3A-C520A/C524A mutation (I) in 293T cells treated with 5 μg/mL Biotin-As.
Developmental arsenic exposure impairs cognition, directly targets DNMT3A and reduces DNA methylation (2022)
Figure 3

Figure 3 - Arsenic induces DNMT3A degradation via the ubiquitin-proteasome pathway.

A Immunoblotting of DNMT3A protein levels in MEFs (upper panel) and CCRF-CEM cells (lower panel) treated with ATO in the indicated conditions.

B MEFs (upper panel) and CCRF-CEM cells (lower panel) were pretreated with 20 μM DMSO, 1 μM MG132, 0.02 μM Concanamycin A (Conc A), 20 μM Calpeptin (Calp), or 20 μM Z-VAD-FMK (Z-VAD) for 2 hours, followed by 16-hour ATO co-treatment at 1 μg/mL for MEFs or 0.3 μg/mL for CCRF-CEM cells. Immunoblotting of DNMT3A in whole cell lysate is shown.

C HCT116 cells were pretreated with 3 μM MG132 for 2 hours, followed by ATO co-treatment for 16 hours at the indicated concentrations. Immunoblotting of DNMT3A in whole cell lysate is shown.

D In vivo ubiquitination assay. 293T cells were transiently transfected with HA-ubiquitin and Myc-DNMT3A. After 24 hours, cells were treated with or without MG132 for 2 hours at the indicated concentrations, followed by ATO co-treatment for 6 hours at the indicated concentrations. Myc-DNMT3A was immunoprecipitated (IP) with anti-Myc antibody, followed by immunoblotting (IB) as indicated.

E Immunoblotting of the transfected wild-type DNMT3A, C520A/C524A and C541A mutants in 293T cells upon 1 μg/mL ATO treatment for the indicated durations.

F Measurement of DNMT3A-ADD thermodynamic stability upon ATO or AsPh3 treatment. Purified DNMT3A-ADD was mixed with ATO or AsPh3 at the indicated ratios in 20 mM HEPES (pH 7.5) for 1 hour. Chemical structure of AsPh3 is shown. Melting curves were measured by DSF (upper panel), and the calculated ΔTm values are shown (lower panel). Data are presented as mean ± SD of n = 4 technical replicates. ns, not significant; ****p < 0.0001 by unpaired two-tailed Student's t test.

G Immunoblotting of DNMT3A in MEFs (upper panel) and CCRF-CEM cells (lower panel) treated with ATO or AsPh3 in the indicated conditions.

H, I Immunoblotting of endogenous DNMT3A, DNMT3B and DNMT1 protein levels in CCRF-CEM (H) and THP-1 (I) cells treated with DAC or ATO in the indicated conditions. DAC: +, 0.2 μM. ATO: +, 0.06 μg/mL; ++, 0.2 μg/mL; +++, 0.6 μg/mL. Red asterisk indicates the specific band.
Developmental arsenic exposure impairs cognition, directly targets DNMT3A and reduces DNA methylation (2022)
Figure 4

Figure 4 - Arsenic induces genome-wide DNA hypomethylation in MEFs.

A Dot blot analysis of global DNA methylation levels in MEFs upon ATO or DAC treatment at the indicated concentrations for 7 days. The relative 5meC levels were quantified by ImageJ. The full blotting image of Fig 4A is shown in Fig EV4D, the treatment is the same.

B COBRA-IAP of global DNA methylation levels in MEFs upon ATO or DAC treatment at the indicated concentrations for 7 days. The signal density of unmethylated and methylated DNA were quantified by ImageJ.

C 5meC enrichment analysis of the IAP, Xist, H19 loci in MEFs upon ATO treatment at 0.3 μg/mL for 4 or 7 days as measured by MeDIP-qPCR. Data are presented as mean ± SD of n = 3 technical replicates. *p < 0.05; **p < 0.01; ***p < 0.001 by unpaired two-tailed Student's t test.

D Dot blot analysis of global DNA methylation levels in CCRF-CEM cells upon ATO or DAC treatment at the indicated concentrations for 7 days. The relative 5meC levels were quantified by ImageJ.

E COBRA-LINE-1 of global DNA methylation levels in CCRF-CEM cells upon ATO or DAC treatment in the indicated conditions. The signal density of unmethylated and methylated DNA were quantified by ImageJ.

F 5meC enrichment analysis of the XIST, H19 loci in CCRF-CEM cells upon ATO treatment at the indicated concentrations for 63 days as measured by MeDIP-qPCR. Data are presented as mean ± SD of n = 3 technical replicates. ns, not significant by unpaired two-tailed Student's t test.
β-catenin activity induces an RNA biosynthesis program promoting therapy resistance in T Acute Lymphoblastic Leukemia (2022)
Figure 1

Figure 1. β-catenin chromatin binding in T-ALL cell lines.

a) Representative density plot (upper panels) and heatmaps (bottom panels) showing β-catenin chromatin binding centered at TSS ± 10 Kb in RPMI8402 or Jurkat cells after precipitation in the indicated conditions with two different antibodies (R&D or SC). The order in the y-axis represents the closest gene annotated to the β-catenin peak obtained in the basal condition with R&D antibody and confirmed in at least another condition (for the RPMI8402 cell line: n=5 for R&D antibody in basal conditions, n =2 for R&D antibody upon LiCl treatment, n=2 for SC antibody in basal conditions, n=2 for SC antibody upon LiCl treatment. For Jurkat cell line: n=3 for R&D antibody in basal conditions in the Jurkat cell line).

b) Genomic distribution of β-catenin ChIPseq peaks shown in a).

c) DNA motifs enriched in the β-catenin binding regions using as an input the peaks obtained in the basal condition with R&D antibody and confirmed in at least another condition.

d) Representative heatmap showing the enrichment of 10Kb upstream and downstream of the identified β-catenin peaks obtained by precipitating TCF1 (n=3), LEF1 (n=3) and Kaiso (n=3) (from left to right).

e) Venn diagram (left panel) representing the number of β-catenin, TCF1/LEF1 and Kaiso targets obtained from ChIPSeq. Each gene dataset is formed by the genes obtained in at least two different replicates for TCF1, LEF1 and Kaiso. Summary of number of peaks and their corresponding annotated genes (right panel) obtained from ChIPSeq of β-catenin, TCF1, LEF1 and Kaiso.

f) Integrative genomic viewer snapshot showing the position of β-catenin, TCF1, LEF1 and Kaiso peaks near the TSS of the indicated genes.

g-h) Representative β-catenin ChIP-qPCR of indicated target genes in wt and TCF1/LEF1 long isoform double KO (DLKO) clones (g) and Kaiso KO clones (h) in RPMI8402 cells. Enrichment of each ChIP is normalized to its respective negative IgG control, indicated by a red-dashed line. DLKO1, DLKO2, KKO1 and KKO2 designate independent single-cell derived clones. Graph represents the mean and sd of 3 technical replicates.
β-catenin activity induces an RNA biosynthesis program promoting therapy resistance in T Acute Lymphoblastic Leukemia (2022)
Figure 2

Figure 2. β-catenin-regulated transcriptional program in T-ALL cell lines.

a) MA plot showing the log2 fold changes for each gene over the mean of normalized counts obtained in the RNAseq analysis of sh-β-catenin vs sh-control (n=3 for each condition). Dots in blue are those genes differentially expressed with an adjusted p-value<0.05.

b) Heatmap showing the differentially expressed genes (adjusted p-value<0.05) for each condition.

c) Biological functions significantly enriched (adjusted p-value < 0.05) in groups of genes downregulated (Down) or upregulated (Up) in the RNAseq analysis.

d) Venn diagram with differentially expressed genes downregulated (DEGs Down) or upregulated (DEGs UP) in the RNAseq analysis compared to the genes obtained in the β-catenin ChIPseq analysis obtained in the basal condition with R&D antibody and confirmed in at least another condition (ChIP targets).

e) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell line (right panel) after knockdown of β-catenin (left panel) compared to control. Graph represents the mean and sd of 6 independent experiments.

f) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell line after treatment with the β-catenin inhibitor ICG-001 (10µM, overnight). Graph represents the mean and sd of 3 independent experiments.

g) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell lines (right panel) after knockdown of ZBTB33/Kaiso (left panel) compared to control. Graph represents the mean and sd of 3 independent experiments.

h-i) Representative flow cytometry histograms of EU incorporation into RNA (h) or OPP incorporation into nascent peptides (i) in the indicated conditions (upper panels). Quantification of relative EU or OPP incorporation. Graph represents the mean and sd of 3 independent experiments (bottom panels). Cells were pre-treated with FH353 30µM or ICG-001 10µM (or DMSO) overnight prior to EU or OPP incubation.

Data information: Statistical significance was determined by two-sided Student's t test.
β-catenin activity induces an RNA biosynthesis program promoting therapy resistance in T Acute Lymphoblastic Leukemia (2022)
Figure 5

Figure 5. β-catenin activity determines cell recovery after chemotherapy treatment in T-ALL cells.

a) Effect of Vincristine (VCR) treatment on cell viability of RPMI8402 cells transduced with sh β-catenin or sh-control. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. *** p ≤ 0.001 of IC50 comparison with respect to sh control

b-c) Effect of VCR (b), Methotrexate (MTX) (c left), L-Asparaginase (L-ASP) (c central) or Cytarabine (Ara-C) (c right) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 comparison with respect VCR-treated cells.

d) Schematic diagram of the experimental approach used for the drug recovery experiments in e) and f). Figure partially created with Biorender.com

e) Cell viability of RPMI8402 cells as fold change relative to day 0 (n = 3). VCR, 15nM; ICG-001, 10 µM. **** p ≤ 0.0001 with respect to control; ### p ≤ 0.001 with respect to VCR-treated cells.

f) Percentage of viability of RPMI8402 cells after drug wash-out during recovery in the presence or absence of ICG-001 10 µM at the indicated time points. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 with respect to recovery in complete medium.

g) Schematic diagram of the experimental approach used for the drug recovery experiments in h) and i). Figure partially created with Biorender.com

h) Cell viability of RPMI8402 cells transduced with sh β-catenin or sh-control as fold change relative to day 0 (n = 3). **** p ≤ 0.0001 untreated sh β-catenin with respect to untreated sh control; #### p ≤ 0.0001, VCR 1nM sh β-catenin with respect to VCR 1nM sh control; ++++ p ≤ 0.0001 , VCR15nM sh β-catenin with respect to VCR 15nM sh control.

i) Percentage of viability of RPMI8402 cells upon β-catenin knockdown after drug wash-out as compared to control at the indicated time points. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, with respect to sh control.

Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for a, b, c, f. i, and h (at day 0), or by one-way ANOVA with Tukey's correction for multiple comparison testing for e and h (at day 2). Exact p-values are disclosed in the Source data file.
β-catenin activity induces an RNA biosynthesis program promoting therapy resistance in T Acute Lymphoblastic Leukemia (2022)
Figure 6

Figure 6. β-catenin inhibition enhances chemotherapy response in a mouse model of T-ALL.

a) Experimental design of in vivo treatment of NSG mice transplanted with RPMI8402 cells. Induction-like chemotherapy: V (vincristine, 0.15 mg·kg-1·day-1), D (dexamethasone, 5 mg·kg-1·day-1), L (L-Asparaginase, 1,000 IU·kg-1·day-1). ICG-001: 10 mg kg-1·day-1.

b) Percentage of RPMI8402 cells in PB at the indicated time points monitoring leukemic progression in the mouse model. RPMI8402 cells identified as human CD45+HLA-ABC+CD5+ by flow cytometry and gated on DAPI negative cells.

c) Percentage of RPMI8402 cells PB, BM, spleen and liver at sacrifice. RPMI8402 cells identified as human CD45+HLA-ABC+CD5+ by flow cytometry and gated on DAPI negative cells.

d-e) Photograph of the livers (d) and spleen (e) collected at sacrifice with the indicated treatments. One control (liver #1 in Fig. 6g) and one Chemo + DMSO (liver #4 in Fig. 6g) mice were sacrificed 2 days before the end of the experiment following humane endpoint criteria.

f) Quantification of spleen (left panel) and liver (right panel) weight normalized to the mouse body weight at sacrifice.

g) H&E staining of representative images per each mouse treated as indicated.

h) Percentage of liver area covered by cellular colonies per 10X micrograph. 4 different images were quantified per animal and mean values per animal are represented by dots.

Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4), following scheme and doses depicted in Fig. 6a and methods.

Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.

EXPANDED VIEW FIGURE LEGENDS
CALCOCO1 acts with VAMP-Associated Proteins to mediate ER-phagy. (2020)
Figure 1

Figure 1. CALCOCO1 is degraded by macroautophagy.

A Domain architecture of CALCOCO paralogs showing the SKICH domain, a conserved LIR motif (LVV), coiled-coli regions (CC) and zinc finger domains (ZF).

B-E Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In C and E, endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.

F A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 and immunostained for endogenous GM130. Scale bars are 5 μm and 2 μm (zoomed inset).

G Same cells as in E were left untreated or treated with Baf A1 for 6 hours, and then immunostained for endogenous p62 and LC3B. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.

H CALCOCO1, p62 and LC3B puncta in in the indicated conditions, counted using an automated system. The error bars represent mean±SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001.

I Percentage of co-localization of CALCOCO1 puncta with p62 and/or LC3B in cells treated as indicated. The error bars represent mean±SEM of three independent experiments per condition and over 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001.

J HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.
CALCOCO1 acts with VAMP-Associated Proteins to mediate ER-phagy. (2020)
Figure 2

Figure 2. CALCOCO1 binds directly to ATG8 family proteins with preference for the GABARAP subfamily.

A GST pulldown binding assay of in vitro transcribed/translated 35S-Myc-CALCOCO1 with recombinant GST-tagged ATG8 family proteins. GST and GST fusion proteins were visualized by coomassie brilliant blue staining (bottom panel), and the co-precipitated Myc-CALCOCO1 was detected by autoradiography (upper panel). The numbers below the AR represent % binding in the shown AR .

B GST pulldown assay of transiently transfected Myc-CALCOCO1 from HEK293 cell extracts with recombinant GST-tagged ATG8 family proteins. GST and GST fusions were visualized by Ponceau S staining (bottom panel), and co-precipitated Myc-CALCOCO1 detected by immunoblotting with anti-Myc antibody (upper panel).

C CALCOCO1 deletion constructs used to map the ATG8 interactions. The red X indicate a point mutation or deletion of the LIR motif. Constructs with no or very weak interaction are indicated in orange.

D-H GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.

I GST pulldown assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST-tagged CALCOCO1 constructs. GST and GST fusions were visualized as in A, and co-precipitated GABARAP with anti-GABARAP antibody (upper panel).