pUS28 Activates Pyk2 and RhoA in U373 Glioblastoma Cells. (A) U373 cells were infected with Ad-Trans or Ad-pUS28 for 18 hrs. Cells were stimulated with 40 ng/ml CCL5 (left) or 40 ng/ml CX3CL1 (right) for the indicated times and analyzed via western blot with a phospho-specific Pyk2-Y402 antibody or total Pyk2 antibody. (B) U373 were infected with Ad-Trans, Ad-pUS28 or Ad-pUS28 + Pyk2 WT or F402Y for 18 hrs. Cells were stimulated with 40 ng/ml CCL5 for the indicated times. Lysates were immunoprecipitated with Rhotekin-RBD-GST Agarose and analyzed by western blot for RhoA. Input lysates were analyzed for total RhoA and to confirm adenovirus infection efficiency. The percent active RhoA was quantified via ImageJ densitometry of both IP and total lysate western blots.