pUS28 Signaling Causes Calcium-Dependent Phosphorylation of Pyk2 at Y402. (A) FAK-/- fibroblasts were infected with Ad-Trans only or Ad-Trans+Ad-pUS28 for 18 hrs. Cells were stimulated with 40 ng/ml CCL5 for the indicated times and analyzed via western blot with a phospho-specific Pyk2-Y402 antibody or total Pyk2 antibody. (B) Adenovirus-infected FAK-/- were pre-treated for 30 min with BAPTA-AM to chelate intracellular calcium and then stimulated with 40 ng/ml CCL5 for 5 min. Total cell lysates were analyzed via western blot for phospho-Y402 Pyk2 and total Pyk2. For both experiments, blots were stripped and reprobed to verify pUS28-HA expression. Phospho-specific blots were quantified via densitometry using ImageJ software and are expressed as fold change compared to unstimulated, Ad-Trans infected control.