Fig. 5. Increased p115 staining at the Golgi in TBC1D20-depleted cells. (A) HeLa cells transfected with GFP-tagged dominant-negative Rabs were fixed after 24 hours, and then stained with antibodies to GM130. The number of cells displaying a fragmented Golgi (open bars) or scattered COPII staining (filled bars) was then counted (n>100 per condition), expressed as a percentage, and plotted as a bar graph. (B) HeLa cells transfected with GFP-tagged Rab1, dominant active Rab1^Q67L, and dominant-negative Rab1 N121I or Rab43 T32N were fixed after 24 hours, and then stained with antibodies to GM130 or COPII (red). Rabs were visualized by GFP fluorescence (green), and DNA was stained with DAPI. (C) Cell lysates from HeLa cells expressing GFP-tagged TBC1D20 treated with either control or TBC1D20-specific siRNA duplexes were western blotted for GFP to detect TBC1D20, and -tubulin as a loading control. (D) HeLa cells expressing the GFP-tagged R105A inactive form of TBC1D20 (green) as a marker for the efficiency of RNA interference were treated with either control or TBC1D20-specific siRNA duplexes for 72 hours. The cells were then fixed and stained with antibodies to COPII, p115 or GM130 (all in red). DNA was stained with DAPI (blue). Bars, 10 µm.