Figure 6. VPS13C deficiency decreases... | Rockefeller University Press

Figure 6.

$VPS13C deficiency decreases lysosomal phospho-Rab10 and impairs phospho-Rab10-mediated lysosomal stress response. (A–D) Representative immunoblot showing phospho-Rab10 (pRab10; T73) (A) and total Rab10 (B) protein levels from whole-cell lysates of HEK-293 FT cells and (C and D) relative quantification of pRab10 and total Rab10 protein levels (N = 4). (E) Representative immunoblot of lysosomal enrichment using LysoIP (anti-HA magnetic beads for LAMP1-RFP-3xHA pulldown). Immunoblot showing lysosomal enrichment efficiency using antibodies against HA, LAMP2 as a lysosomal membrane marker, and GCase as a luminal lysosome marker (equal loading of input and LysoIP fraction) (N = 4). (F and G) (F) Representative immunoblot of pRab10 and total Rab10 protein levels from LysoIP and (G) quantification of protein levels from LysoIP fractions (N = 4). (H–K) Representative immunoblot of pRab10 and total Rab10 protein levels in HEK-293 FT cells treated with the lysosomotropic compound CQ (H) (100 µM, 16 h) or pH-independent lysosomal stressor LLOMe (J) (500 µM, 1 h). Relative quantification of pRab10 normalized to total Rab10 protein levels under CQ (I) or LLOMe (K) treatment (N = 6). Data represented as mean ± SEM; unpaired two-tailed t test (C, D, G, I, and K); ns: not significant (D), *P < 0.05 (C and I), **P < 0.01 (G and K). Source data are available for this figure: SourceData F6.$

VPS13C deficiency decreases lysosomal phospho-Rab10 and impairs phospho-Rab10-mediated lysosomal stress response. (A–D) Representative immunoblot showing phospho-Rab10 (pRab10; T73) (A) and total Rab10 (B) protein levels from whole-cell lysates of HEK-293 FT cells and (C and D) relative quantification of pRab10 and total Rab10 protein levels (N = 4). (E) Representative immunoblot of lysosomal enrichment using LysoIP (anti-HA magnetic beads for LAMP1-RFP-3xHA pulldown). Immunoblot showing lysosomal enrichment efficiency using antibodies against HA, LAMP2 as a lysosomal membrane marker, and GCase as a luminal lysosome marker (equal loading of input and LysoIP fraction) (N = 4). (F and G) (F) Representative immunoblot of pRab10 and total Rab10 protein levels from LysoIP and (G) quantification of protein levels from LysoIP fractions (N = 4). (H–K) Representative immunoblot of pRab10 and total Rab10 protein levels in HEK-293 FT cells treated with the lysosomotropic compound CQ (H) (100 µM, 16 h) or pH-independent lysosomal stressor LLOMe (J) (500 µM, 1 h). Relative quantification of pRab10 normalized to total Rab10 protein levels under CQ (I) or LLOMe (K) treatment (N = 6). Data represented as mean ± SEM; unpaired two-tailed t test (C, D, G, I, and K); ns: not significant (D), *P < 0.05 (C and I), **P < 0.01 (G and K). Source data are available for this figure: SourceData F6.

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