Fig. 4: DRAM1 enhances the stability of lysosomal VAMP8.

A The protein levels of STX17, SNAP29, and VAMP8 were examined by WB in 293 T cells. B, C The protein levels of VAMP8 were measured by WB after transfection with different dosages of Flag-DRAM1. C The quantification of relative VAMP8 protein level. The level of VAMP8 were quantified by Image J and normalized to the corresponding GAPDH (set to 1 at 0 h). D Relative expression levels of VAMP8 were detected by quantitative PCR. E Protein levels of STX17, SNAP29, and VAMP8 were examined by WB with CHX treatment in 293 T cells. F The quantification of relative VAMP8 protein by Image J and normalized with GADPH in 293 T cells. G Localization of TMEM192-3xHA fusion protein with lysosomes in HeLa cells. Scale bars: 10 μm. H Schematic of the workflow for the Lyso-lP method. HA-Lyso cells refer to cells expressing 3xHA-tagged TMEM192. I The lysosomal VAMP8 was examined using the Lyso-lP method in 293 T cells. J The level of lysosomal VAMP8 was quantified by Image J and normalized with corresponding HA-TMEM192 (set to 1 in control cells). K, L Protein levels of VAMP8 in cytoplasmic and lysosomes with CHX treatment in Control and DRAM1 KO cells and quantification. The lysosomes fraction was isolated by a lysosome enrichment kit, and the purity of the fractions was also assessed using LAMP1 (Lysosome markers) and GAPDH (Cytoplasmic) antibodies. M The levels of LC3 and p62 were measured by WB with or without co-transfection of Flag-DRAM1 and GFP-VAMP8. N The protein levels of LC3 and p62 were measured by WB with or without co-transfection of Flag-DRAM1 and si-VAMP8 in 293 T cells. Data in (C, D, F, J, L) are presented as mean ± SD, n = 3 independent experiments. Data in (A, B, E, G, I, K, M, N) are representative of three independent experiments. P-values were calculated by two-tailed Student’s t tests (C, F, J, L) or One-way ANOVA with Tukey’s Multiple Comparison posthoc test (adjusted P-values for multiple comparisons) (D). P values are indicated in the figure.