Fig. 5: DRAM1 inhibits the ubiquitination of VAMP8 by CHIP. | Nature Communications

Fig. 5: DRAM1 inhibits the ubiquitination of VAMP8 by CHIP.

From: DRAM1 promotes the stability of lysosomal VAMP8 to enhance autolysosome formation and facilitates the extravasation

Fig. 5

A The protein levels of VAMP8 were measured by WB in Control and DRAM1 KO cells. B Ubiquitination analysis of extracts of Control or sh-DRAM1 293 T cells transfected with the indicated plasmids. C The quantification of ubiquitination of GFP-VAMP8 from immunoprecipitation by GFP antibody. The bands intensity above 43 KDa corresponding to GFP-VAMP8 was quantified by densitometry analysis (using Image J), and band intensity normalized to the GFP bead band intensity (set to 1 in control cells). D The protein levels of VAMP8 were analyzed by WB. E Effect of CHIP on ubiquitination of VAMP8 from extracts of 293 T cells transfected with GFP-VAMP8 together with or without the Flag-CHIP or si-CHIP. F Effect of CHIP on ubiquitination of VAMP8 from extracts of 293 T cells. G The quantification of ubiquitination of GFP-VAMP8 from immunoprecipitation by GFP antibody. H The endogenous protein level of STX17, SNAP29 and VAMP8 were examined by Co-IP. I, J Representative confocal image showing colocalization between VAMP8 and LAMP1 and quantification (n = 11 cells/group). Data are shown as means ± SD representative of two independent experiments. Scale bars: 10 μm. K Workflow of generation of Chip crispant zebrafish in the background of GFP-Lc3 transgenic zebrafish and lysotracker staining. L Schematic representation of the Chip genetic and protein domain architecture and CRISPR target site. M, N Autophagosome fusion with lysosome was detected by Lysotracker staining in zebrafish larvae. Representative confocal images are shown. Scale bars: 10 μm. The percentage of GFP-LC3 punctae colocalizing with LysoTracker was quantified by Image J in a pre-defined region (n = 12 larvae), data are shown as means ± SD representative of two independent experiments. Data in (C, G) are presented as mean ± SD, n = 3 independent experiments. Data in (A, B, D, E, F, H) and (I, M) are representative of three and two independent experiments, respectively. P-values were calculated by two-tailed Student’s t tests (C and N) or One-way ANOVA with Tukey’s Multiple Comparison post hoc test (adjusted P-values for multiple comparisons) (G and J).

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