Figure 3.
Effect of PTPα over-expression on Src in vivo tyrosine phosphorylation and kinase activity. (A) Neo, PTPα793, PTPα802 and PTPα793(CCSS) overexpressor cells were induced (by removal of doxycycline for 20 h), and lysates were immunoblotted with anti-PTPα polyclonal antibody (panel a) or Src was immunoprecipitated from the lysates and aliquots were immunoblotted with either anti-Src monoclonal antibody (panel b) or anti-pTyr mAb (panel c). Over-expression of PTPα793, PTPα802 or PTPα793(CCSS) decreased tyrosine phosphorylation of Src by 76 ± 7% (n = 4), 79 ± 5% (n = 4) or 19 ± 10% (n = 3), respectively. (B) Lysates from the induced cells were immunoblotted with anti-PTPα polyclonal antibody (panel a) or Src was immunoprecipitated from the lysates and portions were immunoblotted with anti-Src monoclonal antibody (panel b), immunoblotted using anti-dephospho-Y527 Src monoclonal antibody, which reacts only with the activated, Tyr527-dephosphorylated form of Src (panel c), or were subjected to in vitro kinase assay in buffer containing [γ-32P]ATP and acid-denatured enolase (panel d). Over-expression of PTPα793, PTPα802 or PTPα793(CCSS) increased the amount of dephospho-Y527 by factors of 3.8 ± 1.0, 3.9 ± 1.1 or 1.6 ± 0.5 (n = 5) and increased Src kinase activity by factors of 4.9 ± 2.4, 5.9 ± 2.9 or 1.7 ± 0.8 (n = 3), respectively. The positions of molecular weight markers (in kDa) are indicated.