Fig. 1. Expression and focal adhesion localization of PYK2 in fibroblasts. (A) Western blot analysis of HEK 293 cell lysates expressing Myc-tagged PYK2 and FAK using antibodies against Myc, FAK (monoclonal, 2A7) and PYK2 (polyclonal, from UBI). (B) Western blot analysis of PYK2 expression in different cell types. Solid and open arrows indicate PYK2 and FAK, respectively. In (A) and (B), cell lysates were resolved on SDS-PAGE, transferred to nitrocellulose and probed with antibodies as indicated. (C) Co-immunostaining of PYK2 with FAK or paxillin in fibroblasts. 10T1/2 (a-f) and fak^-/- fibroblasts (g-l) were immunostained with the polyclonal antibody against PYK2 (UBI) (a,d,g,j) and the monoclonal antibody against paxillin (b,h) or FAK (e,k); c, f, i and l are overlay pictures. Bar, 50 µm. (D) Time course of fibronectin treatment on PYK2 focal adhesion localization. 10T1/2 fibroblasts were trypsinized and re-plated on fibronectin-coated coverslips for indicated time. These cells were then immunostained with the polyclonal antibody against PYK2 (UBI) (a,d,g) and monoclonal antibody against paxillin (b,e,h); c, f and i are overlay pictures. Scale for D same as C.