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. 2017 Jun 28;8:15866. doi: 10.1038/ncomms15866

Figure 1. FLCN is a GAP for Rab7A.

Figure 1

(a) U2OS cells were transiently transfected with empty vector, FLCN WT, Rab7A WT, Rab7A Q67L, Rab7A T22N or combinations, as indicated. Co-purified complexes were detected by immunoblotting with antibodies recognizing FLCN or Rab7A, as indicated. (b) 293T cells were transiently transfected with an empty vector, FLAG-FLCN WT, FLAG FLCN S62/73A, FLAG FLCN S62/73E, HA-Rab7A WT or combinations, as indicated. Co-purified complexes were detected by immunoblotting with antibodies recognizing FLCN or HA (Rab7A), as indicated. (c) U2OS cells were transfected with empty vector, or FLCN WT and HA-GFP-Rab7A. Co-localization was identified by confocal microscopy (yellow, indicated by arrows in the insert corresponding to the region outlined by the white box). Scale bars, 5 μm (upper panel) and 10 μm (lower panel). (d) HA-Rab7A and FLAG-tagged FLCN WT proteins were purified from transfected 293T cells with anti-HA (Rab7A) or anti-FLAG antibodies. The amount of inorganic phosphate released due to GTPase activity was measured by GTPase colorimetric assay kit. GTPase activity was quantified in four independent experiments, and data are represented as mean±s.e.m. Significance (*) was conferred at P<0.05, ANOVA and Tukey’s Multiple Comparison post-tests. (e) Same as in d, except FLCN WT and FLCN K508R (untagged) were immunoprecipitated with an anti-FLCN antibody from the lysates of transfected 293T cells. The data are represented as mean±s.e.m. and were collected in two independent experiments. * indicates statistical significance, P<0.05, ANOVA and Tukey’s Multiple Comparison post-tests. (f) GST-FLCN WT or GST-FLCN C9 mutant proteins were incubated with HA-Rab7A, purified from transfected 293T cells. The GAP activity was measured as in d,e. Data are represented as mean±s.e.m., n=5. * indicates statistical significance, Two-tailed paired t-test, P=0.0037. (g) U2OS cells were transiently transfected with an empty vector, FLCN WT, Rab7A WT, Rab7B or combinations, as indicated. Co-purified complexes were identified by immunoprecipitation followed by immunoblot, as indicated. (h) Same as in g, except U2OS cells were transiently transfected with Rab7B, Rab35 or Rab8A, as indicated. (i) Same as in g, except U2OS cells were transiently transfected with Rab9A.