Fig. 5.

The actin-binding domains of SH2B1β, pTyr-PAK1, and FLNa are required for maximal PRL-induced membrane ruffling. A, A7 and M2 cell lines were cotransfected with GFP-PRLR, mRFP-tagged WT SH2B1β or SH2B1β Δ-Δ mutant, and myc-tagged WT PAK1 or PAK1 Y3F mutant. 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 WT and/or SH2B1β WT or Δ-Δ localization in ruffles was used to count total number of ruffles per transfected cell. Arrows indicate ruffles, and asterisks denote transfected cells. Scale bar, 20 μm. B, Ruffling index as a number of ruffles per cells 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. NS, Not significant; vctr, vector.