Figure S3.
Activation of autophagy signal by ARL6IP1 via interaction with LC3B. (A) Immunofluorescence staining of ARL6IP1 and LC3B in U2OS cells transfected with pEGFP-tagged ARL6IP1 or pEGFP-empty vectors (600×; scale bars: 20 μm). (B) Immunofluorescence co-localization of LC3B puncta against calnexin, VDAC, and GM130 in HeLa cells expressing GFP-LC3B transfected with FLAG-tagged ARL6IP1 (600×; scale bars: 20 μm). (C) Quantification of LC3B puncta per organelle marker positive cell. Data represent mean ± SD of 10 images (**P < 0.01). Antibodies against the calnexin (endoplasmic reticulum), VDAC-1 (mitochondria), and GM130 (Golgi) used as organelle marker proteins. (D and E) HeLa cells were transduced with Ad-ARL6IP1 or Ad-LacZ at MOI 100, and incubated for 6 h in an HBSS medium, before using the Muse Autophagy LC3-antibody-based kit with the MUSE cell analyzer to detect autophagy. Cytofluorimetric plots of LC3B-II level quantified on a Muse cell analyzer. Data presented as mean values of triplicates ± SD (**P < 0.01). (F) Schematic representation of BiFC constructs involving ARL6IP1 and autophagy-related genes. (G) HEK293 cells transiently transfected with FLAG-tagged ARL6IP1 and GST-tagged LC3B or GST empty vectors (5 μg each). Interaction between exogenous ARL6IP1 and LC3B was detected using GST pull-down and western blot analysis. (H) In vitro binding assay was performed with purified 0.5 µg His-tagged ARL6IP1 and 0.5 µg GST-tagged LC3B or GST protein from the E. coli expression system; analysis was performed via GST pull-down and western blotting. (I) Immunoprecipitation experiments of ARL6IP1 and LC3B from ReNcell CX. (J) Sequence alignment of human LC3/GABARAP proteins. (K) In vitro binding assay between ARL6IP1 and ATG8 family proteins purified from E. coli expression system. All experiments were performed by three independent biological replicates. Source data are available for this figure: SourceData FS3.

Activation of autophagy signal by ARL6IP1 via interaction with LC3B. (A) Immunofluorescence staining of ARL6IP1 and LC3B in U2OS cells transfected with pEGFP-tagged ARL6IP1 or pEGFP-empty vectors (600×; scale bars: 20 μm). (B) Immunofluorescence co-localization of LC3B puncta against calnexin, VDAC, and GM130 in HeLa cells expressing GFP-LC3B transfected with FLAG-tagged ARL6IP1 (600×; scale bars: 20 μm). (C) Quantification of LC3B puncta per organelle marker positive cell. Data represent mean ± SD of 10 images (**P < 0.01). Antibodies against the calnexin (endoplasmic reticulum), VDAC-1 (mitochondria), and GM130 (Golgi) used as organelle marker proteins. (D and E) HeLa cells were transduced with Ad-ARL6IP1 or Ad-LacZ at MOI 100, and incubated for 6 h in an HBSS medium, before using the Muse Autophagy LC3-antibody-based kit with the MUSE cell analyzer to detect autophagy. Cytofluorimetric plots of LC3B-II level quantified on a Muse cell analyzer. Data presented as mean values of triplicates ± SD (**P < 0.01). (F) Schematic representation of BiFC constructs involving ARL6IP1 and autophagy-related genes. (G) HEK293 cells transiently transfected with FLAG-tagged ARL6IP1 and GST-tagged LC3B or GST empty vectors (5 μg each). Interaction between exogenous ARL6IP1 and LC3B was detected using GST pull-down and western blot analysis. (H) In vitro binding assay was performed with purified 0.5 µg His-tagged ARL6IP1 and 0.5 µg GST-tagged LC3B or GST protein from the E. coli expression system; analysis was performed via GST pull-down and western blotting. (I) Immunoprecipitation experiments of ARL6IP1 and LC3B from ReNcell CX. (J) Sequence alignment of human LC3/GABARAP proteins. (K) In vitro binding assay between ARL6IP1 and ATG8 family proteins purified from E. coli expression system. All experiments were performed by three independent biological replicates. Source data are available for this figure: SourceData FS3.

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