Figure 4. HEK293 clones with increased IRS4 expression have elevated PI3K signalling.

(A) Clones of HEK293 cells expressing different levels of IRS4 (#1-low, #2- and #3-high) obtained after retroviral infection and puromycin selection as described in materials and methods, were serum starved and then stimulated with IGF1 or 10% FBS. Cell lysates (30 µg) were subject to immunoblotting with the indicated antibodies. (B) As in (A), except that cells were starved of amino acids and then stimulated with amino acids or 10% FBS for 20 min. (C and D) The cells expressing different levels of IRS4 were serum deprived overnight and assayed for the catalytic activity of immunoprecipitated endogenous Akt and S6K1 using Crosstide peptide as substrate, as described in materials and methods. (E) Clones #1 and #3 of HEK293 cells were serum starved overnight and assayed for PIP₃ as in [37]. Cells from clone #1 were serum starved and treated with IGF1+/− PI-103 (1 µM, 30 min) and used as positive and negative controls, respectively. (F) Cells expressing low (#1) or high (#2 and #3) levels of IRS4 were transfected with control, IRS4 or IRS1 siRNA oligos and the activity of PI3K pathway components was analysed as in (A) under serum starved conditions.