Fig. 1. Characterization of p120 Y228 phosphospecific monoclonal antibody. (A) Detection of phosphorylated p120 on whole cell lysate western blots. 3T3 cells with or without expression of transforming Src 527F were washed for 2 minutes with PBS containing pervanadate, then lysed with RIPA containing pervanadate. Whole cell lysates were separated by SDS-PAGE on a 7% gel and western blotted with mAb pY228 to phosphorylated p120, mAb pp120 to total p120, and mAb PY20, a general phosphotyrosine antibody. The entire blot is shown. (B) mAb pY228 detects p120 by immunoprecipitation. p120, Y228-phosphorylated p120, or various tyrosine phosphorylated proteins were immunoprecipitated from Src-transformed 3T3 cell lysate with mAb 15D2, mAb pY228, or PY20, respectively. Immunoprecipitation with HA-tag antibody 12CA5 served as a negative control. Immunoprecipitates were then western blotted with either mAb pY228, pp120, or PY20. The entire blots are shown for the pY228 and PY20 panels. (C) mAb pY228 is specific for phosphorylated p120. p120 was immunoprecipitated from A431 cells with p120 mAb 15D2. Immunoprecipitates were treated with or without lambda protein phosphatase (New England Biolabs) for 30 minutes at 30°C. Samples were western blotted with mAb pY228 or pp120. (D) mAb pY228 is specific for p120 phosphorylated at Y228. Cos-7 cells were transiently co-transfected with transforming Src (RcRSV c-Src 527F) together with either empty RcRSV vector (lane 1), or RcRSV vector containing mp120-1A (lane 2), mp120-1A/8F (lane 3), or mp120-1A/228F (lane 4), using Superfect reagent (Qiagen). Transfected p120 was specifically immunoprecipitated with murine-specific p120 mAb 8D11, and western blotted with mAb pY228, pp120 or PY20.