Figure 4.

Maximal migration of T47D cells in response to PRL requires tyrosyl phosphorylation of PAK1. A, Equal amount of T47D cells stably overexpressing GFP, PAK1 WT, or PAK1 Y3F were loaded into the upper part of the Boyden chamber. The number of cells that migrated to the lower surface of the chamber toward PRL (black bar) or buffer control (white bar) after 48 h were counted and plotted (B). Scale bar, 300 μm. C and D, T47D cells (C) or MCF-7 cells (D) were transfected with control or PAK1 siRNA, stimulated (black bars) or not (white bars) with PRL, and assessed for migration using the Boyden chamber assay as in panel A. Silencing efficiency was judged by immunoblotting with anti-PAK1 Ab 48 and 72 h after transfection. The expression levels of γ-tubulin (Tu) were used as an internal control (Ctrl). Bars represent mean ± SE. * P < 0.05 compared with the same cells untreated with PRL. n = 3 for each experimental condition.