FIGURE 5:

RGC2 is an endogenous substrate of Akt2 in adipocytes. (A) COS-1 cells were transfected with HA-RGC1 and FLAG-RGC2 constructs as indicated. The RGC proteins were isolated by IP with FLAG antibody and subjected to an in vitro kinase assay with 100 ng recombinant Akt2 kinase and 100 μM ATP for 30 min at 32°C. The immune complexes were resolved on SDS–PAGE and analyzed by WB using the indicated antibodies. (B) 3T3-L1 adipocytes were transfected with control siRNA or siRNA oligos that deplete Akt1, Akt2, or both Akt isoforms. Four days after transfection, serum-starved cells were mock treated or stimulated with 100 nM insulin for 10 min; 2% of total lysates from a 10-cm plate of 3T3-L1 adipocytes were resolved on SDS–PAGE and subjected to WB analysis with the indicated antibodies. (C) Serum-starved 3T3-L1 adipocytes were pretreated with 100 nM wortmannin (WM) for 1 h before stimulation as in (B); 2% of total lysates from a 10-cm plate of 3T3-L1 adipocytes were resolved on SDS–PAGE and analyzed by WB using the indicated antibodies. (D) 293T cells were transfected with HA-RGC1, FLAG-RGC2, and Myr-Akt ΔPH constructs as indicated. Cells were treated with 100 nM WM for 1 h before lysis. RGC2 phosphorylation levels were determined by WB with the RGC2 phospho-715 antibody. (E) Serum-starved 3T3-L1 adipocytes were pretreated with 100 μM Akti-1/2 for 2.5 h before mock treatment or stimulation with 100 nM insulin for 10 min; 2% of total lysates from a 10-cm plate of 3T3-L1 adipocytes were resolved by SDS–PAGE and analyzed by WB using the indicated antibodies.