FIGURE 5.

Effects of pharmacologic inhibition and siRNA knock-down of Hsp90 on AF9. A, IMCD3 cells were treated with Hsp90 inhibitors novobiocin (Novo) and 17AAG as well as siRNA directed against both isoforms of mouse Hsp90. Scrambled siRNA served as a control. Whole cell lysates were prepared, and the relative abundance of proteins was assesses by Western blot. siRNA reduces Hsp90 protein abundance. AF9 protein abundance is not affected by any of the interventions. The vertical lines in the Hsp90 Western blot indicate that the position of the lanes was digitally manipulated. Experiment was repeated in triplicate with similar results. B, IMCD3 cells were treated as in Fig. 5A. Cells were then examined by immunofluorescence microscopy with the antibodies and stains indicated in the figure. AF9 shifts from a primarily nuclear localization to a nuclear and cytoplasmic distribution in cells treated with Hsp90 siRNA or novobiocin. The experiment was repeated in triplicate, and data from one set of experiments are shown. Additional images can be furnished upon request.