Fig. 4: SCFD1 is an autophagic SNARE complex regulator.

A Flag-Trap assays showing the interaction between SCFD1 and VAMP8 upon co-immunoprecipitation (co-IP) using HEK293T cells expressing Flag-SCFD1 and GFP-VAMP8. B Robust interaction between endogenous VAMP8 and Flag-SCFD1. Flag-tagged SCFD1 was transfected into HEK293T cells, Flag-beads were used to pull-down Flag-SCFD1, and VAMP8 was detected by anti-VAMP8 antibody. C Clear signals for Flag-VAMP8 were detected in pull-down analysis of recombinant His-SCFD1 and Flag-VAMP8. D Interaction between transfected Flag-tagged SCFD1 and HA-STX17, observed by Flag co-IP assay. E Robust interaction between Flag-tagged SCFD1 with endogenous STX17, observed in a co-IP assay. STX17 was detected by anti-STX17 antibody. F In vitro pull-down of purified recombinant Flag-STX17 with His-tagged SCFD1, showing the direct interaction between STX17 and SCFD1. G Immunofluorescence under confocal microscopy revealed the colocalization of GFP-SCFD1 with Flag-VAMP8 and endogenous LC3 in U2OS cells under Torin stimulation. Scale bar, 5 μm. H Quantification for Figure G. One-way ANOVA (Data are shown as the mean ± SEM. ****p < 0.0001, n = 20). I The extent of SCFD1/LAMP1 overlap was reduced upon siRNA-mediated knockdown of VAMP8. Immunofluorescence of endogenous LAMP1 and GFP-SCFD1 was observed in U2OS cells with the indicated siRNAs, with Torin treatment. Scale bar, 5 μm. J Quantification for I. One-way ANOVA (Data are shown as the mean ± SEM. ****p < 0.0001, n = 20).