Loss of dynamic ER tubules in Rab18-depleted cells. (a) COS7 cells were treated with control or Rab18.8 3′-UTR siRNA for 52 h, then transfected with empty vector or GFP-Rab18 for 20 h. Cells were fixed and then stained with antibodies to CLIMP-63 and Rtn4. Dotted yellow lines mark the cell boundaries. (b) COS7 cells were treated with control, Rab3GAP (siGAP1+2), Rab18.7, or Rab18.8 3′-UTR siRNA for 52 h, then transfected with empty vector or GFP-Rab18 for 20 h. Cell lysates were Western blotted with antibodies to Rab18, GFP, Rab3GAP subunits, and tubulin as a loading control. Rab18 antibodies see multiple nonspecific bands; lines in the figure indicate endogenous Rab18 and GFP-Rab18. (c) The area of CLIMP-63 as a function of total cell area was measured using ImageJ for 40–60 cells per experiment, for three independent experiments. This was performed for cells expressing (blue bars) or not expressing (green bars) GFP-Rab18. Mean values are plotted in the bar graph, with error bars indicating the standard deviation of the mean. (d) COS7 cells expressing a GFP-ER marker were treated with control or Rab18.7 3′-UTR siRNA for 52 h, then transfected with empty vector or mCherry-Rab18 for 20 h. The cells were then imaged at 2-s intervals using a spinning-disk confocal microscope. The entire cell is shown for t = 0, and a time series showing images every 4 s from the yellow boxed area is depicted in the enlarged regions. Bars are marked in the figure.