pUS28 Signaling Causes Formation of an Active Kinase Complex Involving Pyk2. SMC were infected with Ad-Trans and Ad-Pyk2 +/- Ad-pUS28 and stimulated with 20% serum or 40 ng/ml CCL5 for indicated times. Pyk2 was immunoprecipitated using myc antibodies. In vitro kinase reactions were performed on immunoprecipitated material and reactions were loaded on SDS-PAGE, transferred to immobilon-P membranes and visualized via autoradiography. (A) Autoradiogram of in vitro kinase reactions. Total Pyk2 and pUS28 expression was determined by western blot for myc and HA tags, respectively. (B) Densitometric lane plots of results shown in panel A, generated using ImageJ software. The darker curve (overlay) is unstimulated and the lighter curve (background) is 5 min post-stimulation for Pyk2 only stimulated with serum (top) or Pyk2+pUS28 stimulated with CCL5 (bottom). Black arrows indicate bands present only in stimulated samples. Molecular weight markers are shown on the density plot to facilitate comparison to the autoradiogram in panel A. (C) Overall optical density quantification of each lane in Pyk2 in vitro kinase reactions shown in panel A. Values are displayed as percentages compared to unstimulated samples infected with Ad-Trans+Ad-Pyk2.