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. 2003 Jan 6;160(1):17–23. doi: 10.1083/jcb.200206079

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Copyright © 2003, The Rockefeller University Press

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Figure 1. — p85 interacts with Abi1. (A) Left; lysates (5 mg) from MEFs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (ctr, preimmune serum). Abi1 could also be detected in p85 immunoprecipitates (supplemental materials). About 1% of total p85 could be found associated with Abi1 immunoprecipitates. Right; lysates (5 mg; p85 = 0.3 mg) from MEFs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (top). The immunocomplexes were tested for PI3K lipid kinase activity by TLC (bottom; Domin et al., 1996). (B) Binding of GST-p85 fragments (amino acid boundaries or domains are indicated) to Abi1 from lysates of HA-Abi1–transfected 293T cells. The altered electrophoretic mobility of the Abi1 band in the 320–724 lane is due its co-migration with the GST-fusion protein. (C) Lysates (2 mg) from 293T cells transfected with HA-Abi1 were immunoprecipitated with anti-HA antibody. Immunoprecipitates, treated with alkaline phosphatase in the presence (+) or absence (−) of inhibitors (API), were immunoblotted (IB) with anti-HA (left) or anti-pY (middle), or analyzed in Far-western with the proteins indicated on the top. (D) Lysates (1 mg) from 293T cells, transfected with HA-tagged wild-type (WT) or Y407F Abi1, were subjected to in vitro binding with the GST-NH₂-terminal SH2 domain of p85 (SH2-N) or GST alone. Similar results were also obtained using the COOH-terminal SH2 domain of p85, indicating that the apparent affinities of the SH2 domains of p85 for Abi1 were similar (not depicted). (E) Lysates (5 mg) from Cos-7 cells, transfected with HA-Abi1 (WT) or HA-Abi1Y407F, were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (ctr, anti-flag antibody used as a control).