Constitutive association of tyrosine-phosphorylated proteins with p85/PI3K in HS2 cells.

The position of molecular weight markers are indicated on left and the positions of the different proteins are indicated on right. (A) 663 HS1 cells were either grown in the presence of 2 U/mL Epo or starved in Epo and serum for 4 hours before being stimulated for 30 minutes by 10 U/mL Epo. 606 HS2 cells were grown either with serum or stimulated for 30 minutes with 10 U/mL Epo and serum after 4 hours of serum starvation. Proteins immunoprecipitated from cell lysates (IP) with anti-p85 antibodies were separated by SDS-PAGE and immunoblotted (WB) with anti-PY antibodies. (B) Cell lysates from unstimulated 663 HS1 and 606 HS2 cells were immunoprecipitated with anti–IRS-2, anti-SHIP, anti-Gab1, or anti-p85 antibodies (IP) and analyzed by Western blotting (WB) with antiphosphotyrosine (PY) antibodies or with anti–IRS-2, anti-SHIP, or anti-Gab1 antibodies. (C) Various unstimulated HS2 cell lines were subjected to immunoprecipitation (IP) with anti-p85 antibodies and tyrosine-phosphorylated proteins were detected by Western blotting (WB) with anti-PY antibodies. (D) 606 HS2 cell lysates were immunoprecipitated (IP) with anti-p85, anti-p110α, and anti-p110β antibodies. The immunoprecipitates were subjected to immunoblotting (WB) with anti-p110α, anti-p110β, and anti-Gab1 antibodies (upper panel) or with anti-p85 antibodies (lower panel).