FIGURE 8.

p180 is required for the ER association of AP5. U2OS cells were infected with lentivirus carrying control shRNA (Ctrl shRNA) or shRNAs (clones B9 or B10) against p180. The control and p180-depleted cells were transfected with plasmids containing the ALPP, t-ftz, or AP5 construct and allowed to express mRNAs for 18–24 h. The cells were then treated with either DMSO (C or Ctrl) or HHT for 30 min (H), digitonin-extracted, fixed, stained with specific FISH probes, and imaged. A, representative examples. B, cell lysate collected on the day of injection. The level of depletion was assessed by immunoblotting (WB) against p180 and α-tubulin. C, quantification of the fluorescence intensities of mRNAs in the ER and nucleus. All data were normalized to the ER staining intensity in the control treated/control shRNA group for each construct. Each bar represents the average ± S.E. (error bars) of three independent experiments (each experiment consisting of n>30 cells). Scale bar, 20 μm.