Figure 1.
Increased PI 3-kinase-γ dosage elevates Btk tyrosine phosphorylation in fibroblast cells. Rat-2 cells harboring the SrcE378G mutants infected with retroviruses encoding Btk, Btk, and p101, Btk and p110γ, or Btk with p101 and p110γ were lysed, and Btk protein was immunoprecipitated with anti-Btk antibodies. The immunoprecipitates were subjected to 8% SDS/PAGE and analyzed by immunoblotting. The figure shows the immunoblots incubated with anti-phosphotyrosine antibodies (4G10). The blot was subsequently stripped using Western stripping buffer and probed with polyclonal anti-phosphorylated Tyr-551 (551PYAb), anti-phosphorylated Tyr-223 (223PYAb), or anti-Btk antibody followed by horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies and detected by ECL. A small aliquot (2 × 105 cells) of the whole cell lysate (WCL) was loaded on SDS/PAGE, analyzed for p101 or p120 expression using an anti-myc epitope tag antibody (9E10), followed by horseradish peroxidase-conjugated goat-anti-mouse antibody, and visualized by ECL.