Fig. 7.

Impairment of Pyk2/Grb2 complex formation leads to decreased Shc/Grb2 association within the SHPS-1 signaling complex in response to IGF-I. a Quiescent Pyk2/WT or Pyk2/Y881F expressing SMCs were stimulated with IGF-I. Cell lysates were immunoprecipitated (IP) with anti-Grb2 antibody and immunoblotted (IB) with anti-Shc antibody. The blot was stripped and reprobed with anti-Grb2 antibody. b Lysates from Pyk2/Wt and Pyk2/Y881F mutant cells were immunoprecipitated first with anti-SHPS-1 antibody or normal rabbit IgG. The double-immunoprecipitation was performed as described in “Materials and methods”. After immunoprecipitation of SHPS-1, (I) the SHPS-1 immunoprecipitate was resuspended and immunoprecipitated with anti-Grb2 antibody and immunoblotted (IB) with anti-Pyk2 or anti-Shc antibody; the resultant supernatants after IP (II) with anti-SHPS-1 antibody or normal IgG were immunoprecipitated with anti-Grb2 antibody and immunoblotted (IB) with anti-Shc antibody. The resultant supernatants from same experiment were also directly immunoblotted for β-actin as a loading control. c Lysates from Pyk2/WT and Pyk2/Y881F mutant cells were immunoprecipitated (IP) with anti-pY99 antibody and immunoblotted (IB) with anti-Shc antibody. Twenty micrograms of cell lysate from the same experiment was used for detection of total Shc protein level as a loading control