Fig. 4.

Pyk2 knock-down attenuates Erk1/2 phosphorylation and cell proliferation in response to IGF-I. a SMCs were transduced with LacZ shRNA (control) or Pyk2 shRNA template plasmid and analyzed for Pyk2 protein expression. Cell lysates were immunoblotted with anti-Pyk2 antibody. The blot was stripped and reprobed with anti-β-actin antibody as a loading control. b Twenty micrograms of cell lysate was used for detection of phospho-Erk1/2. The blot was stripped and reprobed with anti-Erk1/2 antibody as a loading control. The protein levels were quantified using scanning densitometry. The graph shows the mean result from three independent experiments expressed as relative pERK/ERK ratio that was calculated from arbitrary scanning units (b, lower panel). c Proliferation of Pyk2/WT or Pyk2/Y881F mutant cells following IGF-I stimulation. Cell proliferation was determined as described in “Materials and methods”. The results represent a mean value (±SE) of six independent experiments. d Quiescent Pyk2 knock-down SMCs were stimulated with IGF-I. Cell lysates were immunoprecipitated (IP) with anti-SHPS-1 antibody and immunoblotted (IB) for Grb2. To control the loading, the blot was stripped and reprobed with anti-SHPS-1 antibody