Fig. 10.

The FLNa-binding domain of SH2B1β, pTyr-PAK1 and FLNa are required for PRL-induced membrane ruffling. A, A7 and M2 cell lines were cotransfected with GFP-PRLR, mRFP-tagged WT SH2B1β (data not shown) or SH2B1β 3Δ mutant (shown), and myc-tagged WT PAK1 (data not shown) or PAK1 Y3F mutant (shown). Cells were treated as in Fig. 3. Myc-PAK1 was visualized by αmyc, whereas PRLR was visualized by GFP fluorescence and mRFP fluorescence for SH2B1β. PAK1 and SH2B1β localization in ruffles was used to count total number of ruffles per transfected cell. Arrows indicate ruffles, and asterisks denote transfected cells. Scale bar, 2 0 μm. B, Ruffling index as a number of ruffles per cell was counted. White bars represent A7 cells, and black bars represent M2 cells. Bars represent mean ± se. *, P < 0.05. Each experiment was repeated three times. n = 300 for each experimental condition. vctr, Vector.