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. 2012 Feb 29;119(17):3951–3961. doi: 10.1182/blood-2011-05-355602

Figure 4.

Figure 4

p85α and p85β differentially regulate signaling in BMMCs. (A) p85α and p85β bind to KIT in response to SCF. Cells (32D) expressing WT KIT and p85α or p85β were starved for 8 hours and stimulated with SCF (100 ng/mL) for 5 minutes. Equal amount of cell lysates (500 μg) were subjected to immunoprecipitation with an anti-KIT antibody followed by Western blot analysis with an anti-HA antibody or immunoprecipitated with an anti-HA antibody followed by Western blot analysis with an anti-KIT antibody. (B) Deficiency of p85β in BMMCs does not impair the expression of p110 catalytic subunits of PI3K. Cell lysates from WT, p85α-, and p85β-deficient BMMCs were subjected to Western blot analysis using antibodies that specifically recognize the indicated forms of p110 catalytic subunits. Expression of various catalytic subunits is indicated. (C) p85α and p85β regulatory subunits of PI3K bind p110α, p110β, and p110δ catalytic subunits with equal efficiency. Cells (32D) expressing p85α or p85β subunit of PI3K were starved and stimulated with SCF and subjected to immunoprecipitation using an anti-HA antibody followed by Western blot analysis with an anti-p110α, anti-p110β, or anti-p110δ antibody. The amount of immunoprecipitated protein in each lane is indicated. (D) Enhanced activation of AKT and ERK MAP kinase in p85β-deficient BMMCs. WT, P85α−/−, and P85β−/− BMMCs were starved overnight in serum- and cytokine-free media and stimulated with SCF (100 ng/mL) for 2 and 5 minutes. Equal amount of protein lysates were subjected to Western blot analysis using an anti–phospho-AKT or anti–phospho-ERK1/2 antibody. Total ERK1/2 protein in each lane is indicated. Similar findings were observed in 2 independent experiments. (E) Deficiency of p85β in BMMCs results in enhanced expression of Mitf. WT, P85α−/−, or P85β−/− BMMCs were lysed, and lysates subjected to Western blot analysis using an anti-Mitf antibody. Expression of Mitf and β-actin in each lane is indicated. A representative Western blot is shown. Similar results were obtained in 3 independent experiments. (F) Overexpression of p85β in WT BMMCs inhibits the expression of Mitf. WT BMMCs expressing empty vector or p85β were lysed and lysates subjected to Western blot analysis using an anti-Mitf antibody. A representative Western blot is shown. Similar results were obtained in 2 independent experiments.