Fig. 1: DRAM1 was identified as VAMP8 interactive partner and induced by starvation. | Nature Communications

Fig. 1: DRAM1 was identified as VAMP8 interactive partner and induced by starvation.

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

Fig. 1

A Endogenous interaction between DRAM1 and VAMP8 as demonstrated with the co-immunoprecipitation (Co-IP) assay in 293 T cells. B The interaction of VAMP8 and DRAM1 was determined by GST pull-down assay. E.coli-expressed GST and GST-VAMP8 were used in GST pull-down assays with lysates of 293 T cells transfected with Flag-DRAM1. C, D Colocalization of VAMP8 with DRAM1 in normal and starvation conditions. Representative confocal images are shown, and the intensity of VAMP8 colocalized with DRAM1 was quantified by Image J (n = 20 cells/group). Scale bars: 10 μm. Data are shown as means ± SD representative for three independent experiments. E, F Relative levels of DRAM1 protein immunoprecipitated by VAMP8 in normal and starvation conditions, and quantification of relative DRAM1 protein level. The level of co-precipitated DRAM1 was quantified by Image J and normalized to the corresponding VAMP8 (set to 1 in 0 h). G Schematic representation of the developmental features of early zebrafish larvae used in autophagy activation assay. H Level of basal autophagy in zebrafish larvae at the indicated time points. Protein samples were extracted from 3, 5, 7, and 10 dpf zebrafish larvae (n = 30 larvae/sample). I Relative expression level of p62, Lc3 and Dram1 at indicated time points. The RNA samples were extracted from 3, 5,7 and 10 dpf zebrafish larvae (n = 30 larvae/sample). J Level of DRAM1 and autophagy activity in 293 T cells under the EBSS treatment at the indicated time points. K The workflow of the experiments shown in L examines the degradation of accumulating Lc3-II in zebrafish larvae after CQ treatment. L Level of p62 and Lc3 protein in WT and mCherry-Dram1 expressing 5 dpf larvae. Protein samples were extracted from zebrafish larvae (n = 30 larvae/sample). Data in (F,I) are presented as mean ± SD, n = 3 independent experiments. Data in (A, B, C, E, H, J, L) are representative of three independent experiments. P values were calculated by two-tailed Student’s t tests (D, F). P-values are indicated in the figure.

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