Journal: Nature Communications

Article Title: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi: 10.1038/s41467-025-64097-4

Figure Lengend Snippet: a A schematic diagram showing the domain organizations of FIP200, ATG16L1, and mammalian ATG8 family proteins. In this drawing, the interactions of ATG16L1 with FIP200 and mammalian ATG8 family proteins are highlighted and indicated by two-way arrows. b Size-exclusion chromatography-based analysis of the interaction of FIP200 Claw with Trx-tagged ATG16L1(78–247). In this panel, the “Sum” stands for the theoretical sum of Trx-tagged ATG16L1(78–247) and FIP200 Claw profiles, while “Mixture” stands for the Trx-tagged ATG16L1(78–247) and FIP200 Claw mixture sample. c Multi-angle light-scattering analysis of the purified ATG16L1(78–247)/FIP200 Claw complex showing the relative light-scattering signals as a function of elution volume. The molecular mass error is the fitted error obtained from the data analysis software. d Sequence alignment analysis of the FIR of ATG16L1 with the currently known FIP200 Claw-binding regions of NAP1, SINTBAD, CCPG1, NDP52, p62, NBR1, Optineurin, and TNIP1 from the human species. In this alignment, the highly conserved acidic residues (Asp, Glu, or potentially phosphorylated Ser residue) and the following two conserved hydrophobic residues are boxed and highlighted with black triangles and stars, respectively. e The fluorescence polarization (FP)-based assay measuring the binding affinity of FIP200 Claw with FITC-labeled ATG16L1 FIR. The K d value is the fitted dissociation constant with standard errors obtained by using the one-site binding model to fit the FP data. f Size-exclusion chromatography-based analysis of the interaction of FIP200 Claw with Trx-tagged ATG16L1(78–235). In this panel, the “Sum” stands for the theoretical sum of Trx-tagged ATG16L1(78–235) and FIP200 Claw profiles, while “Mixture” stands for the Trx-tagged ATG16L1(78–235) and FIP200 Claw mixture sample.

Article Snippet: The EGFP-tagged GABARAPL1(1-115), mEGFP-tagged FIP200, and Flag-tagged ATG16L1 were detected by western blot using the anti-GFP (Proteintech, 50430-2-AP, 1:1000 dilution), anti-GFP (Proteintech, 66002-1-Ig, 1:2000 dilution), anti-Flag (Proteintech, 20543-1-AP, 1:1000 dilution), and anti-Flag (Proteintech, 66008-4-Ig, 1:2000 dilution) primary antibodies.

Techniques: Size-exclusion Chromatography, Multi-Angle Light Scattering, Purification, Software, Sequencing, Binding Assay, Residue, Fluorescence, FP Assay, Labeling

Journal: Nature Communications

Article Title: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi: 10.1038/s41467-025-64097-4

Figure Lengend Snippet: a Ribbon diagram showing the overall structure of the dimeric FIP200 Claw/ATG16L1 FIR complex. In this drawing, two FIP200 Claw molecules are colored in orange and marine, while two bound ATG16L1 FIR motifs are colored in magenta and hot pink, respectively. b Ribbon representation showing the structural comparison of apo -form FIP200 Claw dimer (green, PDB ID: 6DCE ) with the FIP200 Claw/ATG16L1 FIR complex (blue/magenta). In this drawing, the two dimeric structures are overlaid by aligning selected one FIP200 Claw monomer in these two structures. c Ribbon-stick model showing the detailed interactions between FIP200 Claw and ATG16L1 FIR. The hydrogen bonds and salt bridges involved in the interaction are shown as dotted lines. d The combined surface charge representation and the ribbon-stick model showing the charge-charge interactions between FIP200 Claw and ATG16L1 FIR in the solved complex structure.

Article Snippet: The EGFP-tagged GABARAPL1(1-115), mEGFP-tagged FIP200, and Flag-tagged ATG16L1 were detected by western blot using the anti-GFP (Proteintech, 50430-2-AP, 1:1000 dilution), anti-GFP (Proteintech, 66002-1-Ig, 1:2000 dilution), anti-Flag (Proteintech, 20543-1-AP, 1:1000 dilution), and anti-Flag (Proteintech, 66008-4-Ig, 1:2000 dilution) primary antibodies.

Techniques: Comparison

Journal: Nature Communications

Article Title: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi: 10.1038/s41467-025-64097-4

Figure Lengend Snippet: a ITC-based measurement of the binding affinity of GABARAPL1 with Trx-tagged ATG16L1 FIR. b Ribbon diagram showing the overall structure of the GABARAPL1/ATG16L1 FIR complex. In this drawing, the GABARAPL1 molecule is colored in green, while ATG16L1 FIR in magenta. c The ribbon-stick model showing the detailed interactions between GABARAPL1 and ATG16L1 FIR. The hydrogen bonds and salt bridges involved in the interaction are shown as dotted lines. d The combined surface charge representation and the ribbon-stick model showing the charge-charge interactions between GABARAPL1 and ATG16L1 FIR. e The combined surface representation and the ribbon-stick model showing the hydrophobic interactions between GABARAPL1 and ATG16L1 FIR. In this drawing, ATG16L1 FIR is displayed in the ribbon-stick model, and GABARAPL1 is shown in surface representation, colored by different amino acid types. Specifically, the hydrophobic amino acid residues in the surface model of GABARAPL1 are drawn in yellow; the positively charged residues are drawn in blue; the negatively charged residues are drawn in red, and the uncharged polar residues are drawn in gray.

Article Snippet: The EGFP-tagged GABARAPL1(1-115), mEGFP-tagged FIP200, and Flag-tagged ATG16L1 were detected by western blot using the anti-GFP (Proteintech, 50430-2-AP, 1:1000 dilution), anti-GFP (Proteintech, 66002-1-Ig, 1:2000 dilution), anti-Flag (Proteintech, 20543-1-AP, 1:1000 dilution), and anti-Flag (Proteintech, 66008-4-Ig, 1:2000 dilution) primary antibodies.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi: 10.1038/s41467-025-64097-4

Figure Lengend Snippet: a Size-exclusion chromatography coupled with SDS-PAGE analysis of the Trx-ATG16L1(78–247)/GST-GABARAPL1 complex incubated with increasing molar ratio of FIP200 Claw proteins. b The SDS-PAGE combined with Coomassie-blue staining analyses of the protein components of the indicated “fraction 1” and “fraction 2” fractions collected from the analytical gel filtration chromatography experiment at different molar ratios of FIP200 Claw in ( a ). c , d Size-exclusion chromatography coupled with SDS-PAGE analysis of Trx-tagged ATG16L1 FIR wild-type with GABARAPL1 or FIP200 Claw. In this panel, “Sum” stands for the theoretical sum of Trx-tagged ATG16L1 FIR wild-type and GABARAPL1 or FIP200 Claw profiles, while “Mixture” stands for the Trx-tagged ATG16L1 FIR wild-type and GABARAPL1 or FIP200 Claw mixture sample. e , f Size-exclusion chromatography coupled with SDS-PAGE analysis of Trx-tagged ATG16L1 FIR D239R/I240F mutant with GABARAPL1 or FIP200 Claw. In this panel, “Sum” stands for the theoretical sum of Trx-tagged ATG16L1 FIR D239R/I240F mutant and GABARAPL1 or FIP200 Claw profiles, while “Mixture” stands for the Trx-tagged ATG16L1 FIR D239R/I240F mutant and GABARAPL1 or FIP200 Claw mixture sample. g , h Size-exclusion chromatography coupled with SDS-PAGE analysis of Trx-tagged ATG16L1 FIR I240Q/I243Q mutant with GABARAPL1 or FIP200 Claw. In this panel, “Sum” stands for the theoretical sum of Trx-tagged ATG16L1 FIR I240Q/I243Q mutant and GABARAPL1 or FIP200 Claw profiles, while “Mixture” stands for the Trx-tagged ATG16L1 FIR I240Q/I243Q mutant and GABARAPL1 or FIP200 Claw mixture sample.

Article Snippet: The EGFP-tagged GABARAPL1(1-115), mEGFP-tagged FIP200, and Flag-tagged ATG16L1 were detected by western blot using the anti-GFP (Proteintech, 50430-2-AP, 1:1000 dilution), anti-GFP (Proteintech, 66002-1-Ig, 1:2000 dilution), anti-Flag (Proteintech, 20543-1-AP, 1:1000 dilution), and anti-Flag (Proteintech, 66008-4-Ig, 1:2000 dilution) primary antibodies.

Techniques: Size-exclusion Chromatography, SDS Page, Incubation, Staining, Filtration, Chromatography, Mutagenesis

Journal: Nature Communications

Article Title: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi: 10.1038/s41467-025-64097-4

Figure Lengend Snippet: a Co-immunoprecipitation assays showing that point mutations of key interface residues of GABARAPL1 observed in the GABARAPL1/ATG16L1 FIR complex structure essentially disrupt their specific interaction in cells. “IB” means immunoblotting. b Co-immunoprecipitation assays showing that point mutations of both devised ATG16L1 mutant and key interface residues of FIP200 observed in the FIP200 Claw/ATG16L1 FIR complex structure decrease or essentially disrupt the specific interaction between FIP200 and ATG16L1 in cells. c Western blot-based measurements of the LC3B lipidation and p62 degradation levels in ATG16L1 -knockout HeLa cells (16KO) as well as that rescued with mEGFP-tagged WT ATG16L1 (16KO + WT), ATG16L1 D239R/I240F mutant (16KO + DRIF), or ATG16L1 I240Q/I243Q mutant (16KO + IQIQ) treated for 4 h using D10 + PS normal medium (F), D10 + PS normal medium treated with bafilomycin A1 at 400 nM (F + B), amino acid starvation medium (S), or amino acid starvation medium treated with bafilomycin A1 at 400 nM (S + B). d The levels of p62 and β-actin in ( c ) were quantified in ImageJ and normalized to that of 16KO cells treated with S + B. The data is presented as means ± SEM from four independent experiments. e The levels of LC3B-II and β-actin in ( c ) were quantified in ImageJ and normalized to the 16KO + WT cells treated with S + B. The data is presented as means ± SEM from four independent experiments. f Western blot-based measurements of the LC3B lipidation in ATG16L1 -knockout HeLa cells (16KO) as well as that rescued with mEGFP-tagged WT ATG16L1 (16KO + WT), ATG16L1 D239R/I240F mutant (16KO + DRIF), ATG16L1 I240Q/I243Q mutant (16KO + IQIQ), ATG16L1 K490A mutant (16KO + K490A), ATG16L1 K490A/D239R/I240F mutant (16KO + K490A/DRIF), or ATG16L1 K490A/I240Q/I243Q mutant (16KO + K490A/IQIQ) treated for 1 h using D10 + PS normal medium (F), D10 + PS normal medium treated with monensin at 100 μM (M), or D10 + PS normal medium treated with monensin at 100 μM and bafilomycin A1 at 400 nM (M + B). g The levels of LC3B-II and β-actin in ( f ) were quantified and normalized to that of 16KO + DRIF cells treated with monensin at 100 μM (M). The data are presented as means ± SEM from three independent experiments. Statistical analyses were all performed in GraphPad Prism 9 by two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test, and P value style is P = 0.1234 [not significant (ns)], *P = 0.0332, **P = 0.0021, ***P = 0.0002, and ****P < 0.0001. h A proposed model depicting the functions of the FIP200/ATG16L1 and ATG8s/ATG16L1 interactions in canonical autophagy.

Article Snippet: The EGFP-tagged GABARAPL1(1-115), mEGFP-tagged FIP200, and Flag-tagged ATG16L1 were detected by western blot using the anti-GFP (Proteintech, 50430-2-AP, 1:1000 dilution), anti-GFP (Proteintech, 66002-1-Ig, 1:2000 dilution), anti-Flag (Proteintech, 20543-1-AP, 1:1000 dilution), and anti-Flag (Proteintech, 66008-4-Ig, 1:2000 dilution) primary antibodies.

Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Knock-Out

Journal: Nature Communications

Article Title: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi: 10.1038/s41467-025-64097-4

Figure Lengend Snippet: a A schematic diagram showing the domain organizations of FIP200, ATG16L1, and mammalian ATG8 family proteins. In this drawing, the interactions of ATG16L1 with FIP200 and mammalian ATG8 family proteins are highlighted and indicated by two-way arrows. b Size-exclusion chromatography-based analysis of the interaction of FIP200 Claw with Trx-tagged ATG16L1(78–247). In this panel, the “Sum” stands for the theoretical sum of Trx-tagged ATG16L1(78–247) and FIP200 Claw profiles, while “Mixture” stands for the Trx-tagged ATG16L1(78–247) and FIP200 Claw mixture sample. c Multi-angle light-scattering analysis of the purified ATG16L1(78–247)/FIP200 Claw complex showing the relative light-scattering signals as a function of elution volume. The molecular mass error is the fitted error obtained from the data analysis software. d Sequence alignment analysis of the FIR of ATG16L1 with the currently known FIP200 Claw-binding regions of NAP1, SINTBAD, CCPG1, NDP52, p62, NBR1, Optineurin, and TNIP1 from the human species. In this alignment, the highly conserved acidic residues (Asp, Glu, or potentially phosphorylated Ser residue) and the following two conserved hydrophobic residues are boxed and highlighted with black triangles and stars, respectively. e The fluorescence polarization (FP)-based assay measuring the binding affinity of FIP200 Claw with FITC-labeled ATG16L1 FIR. The K d value is the fitted dissociation constant with standard errors obtained by using the one-site binding model to fit the FP data. f Size-exclusion chromatography-based analysis of the interaction of FIP200 Claw with Trx-tagged ATG16L1(78–235). In this panel, the “Sum” stands for the theoretical sum of Trx-tagged ATG16L1(78–235) and FIP200 Claw profiles, while “Mixture” stands for the Trx-tagged ATG16L1(78–235) and FIP200 Claw mixture sample.

Article Snippet: Endogenous FIP200, ATG5~ATG12, WIPI2, GABARAP family proteins, and mEGFP-tagged wild-type ATG16L1 or relevant ATG16L1 mutants were detected by western blot using the anti-FIP200 (Proteintech, 17250-1-AP, 1:2000 dilution), anti-ATG5 (Proteintech, 81803-1-RR, 1:2000 dilution), anti-WIPI2 (Proteintech, 28820-1-AP, 1:2000 dilution), anti-GABARAP family (Selleck, F1156, 1:2000 dilution), and anti-GFP (Abmart, M20004M, 1:2000 dilution) primary antibodies.

Techniques: Size-exclusion Chromatography, Multi-Angle Light Scattering, Purification, Software, Sequencing, Binding Assay, Residue, Fluorescence, FP Assay, Labeling

Journal: Nature Communications

Article Title: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi: 10.1038/s41467-025-64097-4

Figure Lengend Snippet: a Ribbon diagram showing the overall structure of the dimeric FIP200 Claw/ATG16L1 FIR complex. In this drawing, two FIP200 Claw molecules are colored in orange and marine, while two bound ATG16L1 FIR motifs are colored in magenta and hot pink, respectively. b Ribbon representation showing the structural comparison of apo -form FIP200 Claw dimer (green, PDB ID: 6DCE ) with the FIP200 Claw/ATG16L1 FIR complex (blue/magenta). In this drawing, the two dimeric structures are overlaid by aligning selected one FIP200 Claw monomer in these two structures. c Ribbon-stick model showing the detailed interactions between FIP200 Claw and ATG16L1 FIR. The hydrogen bonds and salt bridges involved in the interaction are shown as dotted lines. d The combined surface charge representation and the ribbon-stick model showing the charge-charge interactions between FIP200 Claw and ATG16L1 FIR in the solved complex structure.

Article Snippet: Endogenous FIP200, ATG5~ATG12, WIPI2, GABARAP family proteins, and mEGFP-tagged wild-type ATG16L1 or relevant ATG16L1 mutants were detected by western blot using the anti-FIP200 (Proteintech, 17250-1-AP, 1:2000 dilution), anti-ATG5 (Proteintech, 81803-1-RR, 1:2000 dilution), anti-WIPI2 (Proteintech, 28820-1-AP, 1:2000 dilution), anti-GABARAP family (Selleck, F1156, 1:2000 dilution), and anti-GFP (Abmart, M20004M, 1:2000 dilution) primary antibodies.

Techniques: Comparison

Journal: Nature Communications

Article Title: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi: 10.1038/s41467-025-64097-4

Figure Lengend Snippet: a ITC-based measurement of the binding affinity of GABARAPL1 with Trx-tagged ATG16L1 FIR. b Ribbon diagram showing the overall structure of the GABARAPL1/ATG16L1 FIR complex. In this drawing, the GABARAPL1 molecule is colored in green, while ATG16L1 FIR in magenta. c The ribbon-stick model showing the detailed interactions between GABARAPL1 and ATG16L1 FIR. The hydrogen bonds and salt bridges involved in the interaction are shown as dotted lines. d The combined surface charge representation and the ribbon-stick model showing the charge-charge interactions between GABARAPL1 and ATG16L1 FIR. e The combined surface representation and the ribbon-stick model showing the hydrophobic interactions between GABARAPL1 and ATG16L1 FIR. In this drawing, ATG16L1 FIR is displayed in the ribbon-stick model, and GABARAPL1 is shown in surface representation, colored by different amino acid types. Specifically, the hydrophobic amino acid residues in the surface model of GABARAPL1 are drawn in yellow; the positively charged residues are drawn in blue; the negatively charged residues are drawn in red, and the uncharged polar residues are drawn in gray.

Article Snippet: Endogenous FIP200, ATG5~ATG12, WIPI2, GABARAP family proteins, and mEGFP-tagged wild-type ATG16L1 or relevant ATG16L1 mutants were detected by western blot using the anti-FIP200 (Proteintech, 17250-1-AP, 1:2000 dilution), anti-ATG5 (Proteintech, 81803-1-RR, 1:2000 dilution), anti-WIPI2 (Proteintech, 28820-1-AP, 1:2000 dilution), anti-GABARAP family (Selleck, F1156, 1:2000 dilution), and anti-GFP (Abmart, M20004M, 1:2000 dilution) primary antibodies.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi: 10.1038/s41467-025-64097-4

Figure Lengend Snippet: a Size-exclusion chromatography coupled with SDS-PAGE analysis of the Trx-ATG16L1(78–247)/GST-GABARAPL1 complex incubated with increasing molar ratio of FIP200 Claw proteins. b The SDS-PAGE combined with Coomassie-blue staining analyses of the protein components of the indicated “fraction 1” and “fraction 2” fractions collected from the analytical gel filtration chromatography experiment at different molar ratios of FIP200 Claw in ( a ). c , d Size-exclusion chromatography coupled with SDS-PAGE analysis of Trx-tagged ATG16L1 FIR wild-type with GABARAPL1 or FIP200 Claw. In this panel, “Sum” stands for the theoretical sum of Trx-tagged ATG16L1 FIR wild-type and GABARAPL1 or FIP200 Claw profiles, while “Mixture” stands for the Trx-tagged ATG16L1 FIR wild-type and GABARAPL1 or FIP200 Claw mixture sample. e , f Size-exclusion chromatography coupled with SDS-PAGE analysis of Trx-tagged ATG16L1 FIR D239R/I240F mutant with GABARAPL1 or FIP200 Claw. In this panel, “Sum” stands for the theoretical sum of Trx-tagged ATG16L1 FIR D239R/I240F mutant and GABARAPL1 or FIP200 Claw profiles, while “Mixture” stands for the Trx-tagged ATG16L1 FIR D239R/I240F mutant and GABARAPL1 or FIP200 Claw mixture sample. g , h Size-exclusion chromatography coupled with SDS-PAGE analysis of Trx-tagged ATG16L1 FIR I240Q/I243Q mutant with GABARAPL1 or FIP200 Claw. In this panel, “Sum” stands for the theoretical sum of Trx-tagged ATG16L1 FIR I240Q/I243Q mutant and GABARAPL1 or FIP200 Claw profiles, while “Mixture” stands for the Trx-tagged ATG16L1 FIR I240Q/I243Q mutant and GABARAPL1 or FIP200 Claw mixture sample.

Article Snippet: Endogenous FIP200, ATG5~ATG12, WIPI2, GABARAP family proteins, and mEGFP-tagged wild-type ATG16L1 or relevant ATG16L1 mutants were detected by western blot using the anti-FIP200 (Proteintech, 17250-1-AP, 1:2000 dilution), anti-ATG5 (Proteintech, 81803-1-RR, 1:2000 dilution), anti-WIPI2 (Proteintech, 28820-1-AP, 1:2000 dilution), anti-GABARAP family (Selleck, F1156, 1:2000 dilution), and anti-GFP (Abmart, M20004M, 1:2000 dilution) primary antibodies.

Techniques: Size-exclusion Chromatography, SDS Page, Incubation, Staining, Filtration, Chromatography, Mutagenesis

Journal: Nature Communications

Article Title: Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi: 10.1038/s41467-025-64097-4

Figure Lengend Snippet: a Co-immunoprecipitation assays showing that point mutations of key interface residues of GABARAPL1 observed in the GABARAPL1/ATG16L1 FIR complex structure essentially disrupt their specific interaction in cells. “IB” means immunoblotting. b Co-immunoprecipitation assays showing that point mutations of both devised ATG16L1 mutant and key interface residues of FIP200 observed in the FIP200 Claw/ATG16L1 FIR complex structure decrease or essentially disrupt the specific interaction between FIP200 and ATG16L1 in cells. c Western blot-based measurements of the LC3B lipidation and p62 degradation levels in ATG16L1 -knockout HeLa cells (16KO) as well as that rescued with mEGFP-tagged WT ATG16L1 (16KO + WT), ATG16L1 D239R/I240F mutant (16KO + DRIF), or ATG16L1 I240Q/I243Q mutant (16KO + IQIQ) treated for 4 h using D10 + PS normal medium (F), D10 + PS normal medium treated with bafilomycin A1 at 400 nM (F + B), amino acid starvation medium (S), or amino acid starvation medium treated with bafilomycin A1 at 400 nM (S + B). d The levels of p62 and β-actin in ( c ) were quantified in ImageJ and normalized to that of 16KO cells treated with S + B. The data is presented as means ± SEM from four independent experiments. e The levels of LC3B-II and β-actin in ( c ) were quantified in ImageJ and normalized to the 16KO + WT cells treated with S + B. The data is presented as means ± SEM from four independent experiments. f Western blot-based measurements of the LC3B lipidation in ATG16L1 -knockout HeLa cells (16KO) as well as that rescued with mEGFP-tagged WT ATG16L1 (16KO + WT), ATG16L1 D239R/I240F mutant (16KO + DRIF), ATG16L1 I240Q/I243Q mutant (16KO + IQIQ), ATG16L1 K490A mutant (16KO + K490A), ATG16L1 K490A/D239R/I240F mutant (16KO + K490A/DRIF), or ATG16L1 K490A/I240Q/I243Q mutant (16KO + K490A/IQIQ) treated for 1 h using D10 + PS normal medium (F), D10 + PS normal medium treated with monensin at 100 μM (M), or D10 + PS normal medium treated with monensin at 100 μM and bafilomycin A1 at 400 nM (M + B). g The levels of LC3B-II and β-actin in ( f ) were quantified and normalized to that of 16KO + DRIF cells treated with monensin at 100 μM (M). The data are presented as means ± SEM from three independent experiments. Statistical analyses were all performed in GraphPad Prism 9 by two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test, and P value style is P = 0.1234 [not significant (ns)], *P = 0.0332, **P = 0.0021, ***P = 0.0002, and ****P < 0.0001. h A proposed model depicting the functions of the FIP200/ATG16L1 and ATG8s/ATG16L1 interactions in canonical autophagy.

Article Snippet: Endogenous FIP200, ATG5~ATG12, WIPI2, GABARAP family proteins, and mEGFP-tagged wild-type ATG16L1 or relevant ATG16L1 mutants were detected by western blot using the anti-FIP200 (Proteintech, 17250-1-AP, 1:2000 dilution), anti-ATG5 (Proteintech, 81803-1-RR, 1:2000 dilution), anti-WIPI2 (Proteintech, 28820-1-AP, 1:2000 dilution), anti-GABARAP family (Selleck, F1156, 1:2000 dilution), and anti-GFP (Abmart, M20004M, 1:2000 dilution) primary antibodies.

Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Knock-Out