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. 2019 Dec 4;8:e49930. doi: 10.7554/eLife.49930

Figure 7. TBC1D15 reduces GTP-bound Rab7.

Figure 7.

(A) WB and quantification of LC3-II from HeLa cells transfected with scrambled siRNA or siRNA against Rab7 (Rab7Δ) +/- bafilomycin (4 hr, 400 nM). Data are mean fluorescence intensities of bands ± SEM normalised to α-tubulin (n = 3). *p<0.05, ***p<0.001 (two-way ANOVA). (B) WB and quantification of LC3-II normalised to α-tubulin from HeLa cells co-expressing pcDNA3.1 and EGFP, or TBC1D15 with either EGFP, pIRESneo-myc--Rab7wt, EGFP-Rab7Q67L, or EGFP-Rab7T22N for 24 hr before treatment with bafilomycin (4 hr, 400 nM). Data are mean fluorescence intensities of bands ± SEM normalised to α-tubulin (n = 6). *p<0.05, **p<0.01, ***p<0.001 (two-way ANOVA). (C) Immunofluorescence (IF) images of HeLa cells co-expressing TBC1D15 with pIRESneo-myc-Rab7wt, EGFP-Rab7Q67L or EGFP-Rab7T22N stained with antibodies against LC3 and TBC1D15. Scale bars, 10 μm. (D) WB showing immunoprecipitation (IP) of GTP-bound (active) Rab7 from HeLa expressing pcDNA3.1 or TBC1D15. Data are mean fluorescence intensities of GTP-bound Rab7 (IP) normalized to the endogenous level of Rab7 (cell lysate) ± SEM (n = 3). **p<0.01 (Student’s t-test). (E)IF of HeLa cells expressing empty vector or TBC1D15 stained with antibodies against GTP-bound Rab7, TBC1D15 and DAPI. Scale bar, 10 μm.