Figure 3.

FLCN functions as an activator toward Rab35 in vitro. (A) HEK293T cells were transiently transfected with Flag-FLCN, Flag-FLCN-NT, or Flag-FLCN-CT plasmid. At 36 h after transfection, lysates of HEK-293T cells was incubated in vitro with GST-tagged Rab35 or GST alone, and coprecipitation of FLCN with GST fusion Rab35 proteins, bound to glutathione-beads, was analyzed by western blot using an anti-Flag antibody. GST, GST alone used as a control. (B) HEK293T cells were transiently transfected with Rab35 CA, Rab35 WT, or Rab35 DN plasmid. At 36 h after transfection, lysates of HEK-293T cells was incubated in vitro with GST-tagged FLCN-CT or GST alone, and coprecipitation of Rab35 with GST fusion FLCN-CT proteins, bound to glutathione-beads, was analyzed by western blot using an anti-GFP antibody. GST, GST alone used as a control. (C) Equal amounts of lysates from the indicated HeLa cells transiently transfected with Rab35 CA, Rab35 WT, or Rab35 DN plasmid were immunoprecipitated with an anti-GFP antibody or IgG, and then both unprocessed lysates (Input) and immunoprecipitates were resolved by western blot using the indicated antibodies. (D) HEK293T cells were transiently transfected with negative control siRNA or FLCN siRNA pool. At 36 h after transfection, cells were lysed and subjected to GST-pulldown analysis for the activity of Rab35. (E) HEK293T cells were transiently transfected with pCMV-Flag empty vector, Flag-FLCN, Flag-FLCN-NT, or Flag-FLCN-CT plasmid. At 36 h after transfection, cells were lysed and subjected to GST-pulldown analysis for the activity of Rab35.