Figure 1.

Characterization of TMX2–28 Cell Lines Stably Expressing GFP, or GFP with myc-Tagged PAK1 WT and PAK1 Y3F. A, Parental TMX2–28 cells (lane 1), TMX2–28 cells stably expressing either GFP (lane 2), myc-PAK1 WT (lane 3), or myc-PAK1 Y3F (lane 4) were lysed, and proteins were resolved by SDS-PAGE. Overexpressed proteins were visualized by immunoblotting with anti-myc Ab (upper panel). The same polyvinylidene difluoride (PVDF) membrane was stripped and reblotted with anti-PAK1 Ab to visualize endogenous (lanes 1 and 2) and overexpressed PAK1 (lanes 3 and 4) (upper middle panel) and with anti-GFP Ab (low middle panel). Immunoblotting with antiactin Ab was used as a loading control (bottom panel). B, myc-PAK1 was IP'd with anti-myc Ab from TMX2–28 PAK1 WT (lanes 1–3) or TMX2–28 PAK1 Y3F cells (lanes 4–6) transfected with vector (lanes 1 and 4), constitutive active Rac1 V12 (lanes 2 and 5), or constitutive active Cdc42 L61 (lanes 3 and 6) and immunoblotted with the indicated antibodies. The migrations of proteins are indicated. C, TMX2–28 PAK1 WT (left panel) and TMX2–28 PAK1 Y3F cells (right panel) were deprived of serum overnight and treated with or without 30 ng/mL HRG for 15 minutes. Myc-PAK1 was IP'd with anti-myc Ab and subjected to an in vitro kinase assay with H4 histone as a substrate and probed with anti-PAK1 Ab. Relative PAK1 kinase activity was then normalized by the amount of IP'd PAK1 for each lane. The numbers at the bottom of upper and middle panels give the relative fold increase of ³²P incorporation into H4 histone (upper panel) and into PAK1 protein (middle panel). The migrations of proteins are indicated. All blots are representative of at least 3 experiments. D, TMX2–28 PAK1 WT and TMX2–28 PAK1 Y3F cells were deprived of serum overnight and treated with or without 200 ng/mL PRL for 20 minutes. Myc-PAK1 was IP'd with anti-myc Ab and subjected to the in vitro kinase assay as described in panel C. Relative PAK1 kinase activity was then normalized by the amount of IP'd PAK1 for each lane and plotted (right panel). The migrations of proteins are indicated. Bars represent mean ± S.E. *, P < .05 compared with the same cells untreated with PRL (n = 3). All blots are representative of at least 3 experiments.