Figure 6

D609-induced retardation of reexpression of HER2 on the plasma membrane of SKBr3 cells after short-term receptor engagement with an anti-HER2 antibody. (a) CLSM examinations on unfixed cells transiently engaged (30 minutes at 4°C) with α-HER2 mAb 300G9, followed by goat α-mouse Alexa Fluor-594 (panel a, red), then cultured at 37°C for the indicated time periods in complete Ab-free medium, either in the absence (b through g) or presence of D609, 50 μg/mL (h through o). At the end of each time interval, cells were stained again on the plasma membrane with the α-HER2 W6/100 mAb, followed by goat α-mouse Alexa Fluor-488 (green). Nuclei were stained with DAPI (blue). Scale bars, 8 μm. These analyses were independently performed 3 times. (b) Western blot analyses of SKBr3 cells first engaged with α-HER2 300G9 mAb, followed by goat α-mouse antibody, and then cultured at 37°C either in the absence or presence of D609 for the indicated time periods. The blots were incubated with α-HER2 or with anti-β actin Abs. These experiments were repeated on seven sets of independent cell preparations.