FIGURE 7.

p180 is required for the initial ER targeting of ALPP. U2OS cells were infected with lentivirus carrying control shRNA (Ctrl shRNA/Ctrl kd) or shRNA clone B10 against p180 (p180 kd). The control and p180-depleted cells were pretreated with DMSO (Ctrl) or HHT for 15 min, then microinjected with plasmids containing either the ALPP or t-ftz and allowed to express mRNAs for 2 h in the presence of DMSO or HHT. The cells were then extracted with digitonin, fixed, and 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.