Figure 3.

Depletion of Rab14, but not Rab4, abolishes endosomal localization of RUFY1. HeLa cells were treated with a pool of siRNAs for LacZ, Rab14 or both Rab4a and Rab4b. After 72 h, the cells were lysed to be processed for immunoblot analysis using antibodies to indicated proteins (A) or fixed with 3% PFA, permeabilized, and immunostained for Rab14 (B). The siRNA-treated cells were permeabilized with saponin before fixation with PFA and immunostained for Rab14 and RUFY1 using rabbit polyclonal anti-Rab14 and mouse polyclonal anti-RUFY1 antibodies. (C) The number of cells in which RUFY1 localized to endosomes was counted and normalized with total counted cell numbers. The graph is a representative of four independent experiments. (D) HeLa cells treated with siRNAs for Rab14 were transfected with a plasmid encoding HA-Rab14, fixed, and immunostained with anti-RUFY1 and anti-HA antibodies. Bars, 20 μm.