Fig. 6: The amino sites of 68,72,75 of VAMP8 are required for DRAM1 mediated ubiquitination. | Nature Communications

Fig. 6: The amino sites of 68,72,75 of VAMP8 are required for DRAM1 mediated ubiquitination.

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

Fig. 6

A Schematic structure of human VAMP8 and its mutants used in this study. Positions of the SNARE motif, transmembrane domain and the seven Lys residues are indicated. B The ubiquitination of GFP-VAMP8 and its mutants were examined by Co-IP. GFP-tagged VAMP8 and its mutants were transfected into 293 T cells for 48 h, and immunoprecipitated with anti-GFP antibody. C Schematic structure of mutation of human VAMP8 4×K-R and VAMP8 3×K-R used in this study. D The ubiquitination of GFP-VAMP8, VAMP8 4×K-R and VAMP8 3×K-R were examined by Co-IP. GFP-tagged VAMP8, VAMP8 4×K-R and VAMP8 3×K-R were transfected into 293 T cells for 48 h, and cell lysates were immunoprecipitated with anti GFP antibody. Transfected cells were then treated with MG132 (10 μM) for 6 h before collection. E The quantification of ubiquitination of GFP-VAMP8 from immunoprecipitation by GFP. The bands intensity above 43KDa corresponding to GFP-VAMP8 were quantified by densitometry analysis (using Image J), and band intensity normalized to the GFP bead band intensity (set to 1 in control cells). Data are shown as means ± SD from three independent experiments. F The protein level of GFP-VAMP8 in the presence or absence of Flag-CHIP by WB assay. 293 T cells were co-transfected with the indicated vector for 48 h, then, the cell lysate was used for WB analysis. G The effect of CHIP on assembly of STX17-SNAP29-VAMP8 complex in the presence of VAMP83×K-R. 293 T cells were co-transfected with the indicated vector for 48 h, and cell lysates were immunoprecipitated with anti STX17 or SNAP29 antibody, respectively. H The effect of DRAM1 on assembly of STX17-SNAP29-VAMP8 complex in the presence of VAMP83K-R. 293 T cells were co-transfected with the indicated vector for 48 h, and cell lysates were immunoprecipitated with anti STX17 or SNAP29 antibody, respectively. Data in (B, D, F–H) are representative of three independent experiments. P-values were calculated by One-way ANOVA with Tukey’s Multiple Comparison post hoc test (adjusted P-values for multiple comparisons) (E). P-values are indicated in the figure.

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