Figure 5. AKT/β-actin complex is not disturbed by inhibition of AKT phosphorylation.

(A) A2058 cells were serum starved for 24 h and then stimulated (+) or not (−) with 10% serum for 30 min as indicated. Nuclear fractions were immunoprecipitated (IP) with anti-total AKT or control IgG antibodies. Cell lysates prior to immunoprecipitation were used as input. Input and co-IP proteins were resolved on SDS-PAGE and immunoblotted for the detection of p-AKT Ser⁴⁷³, total AKT and β-actin (B). The result shown in (A) was validated by reverse IP where anti-β-actin was used for immunoprecipitation instead of anti-total AKT. (C) A2058 cells were serum starved for 24 h in the presence (+) or absence (−) of the PI3K inhibitor XI (20 μM) and then stimulated (+) or not (−) with 10% serum for 30 min. Nuclear fractions were analyzed by Western blot with anti-p-AKT Ser⁴⁷³, anti-total AKT and anti-β-actin. (D) A2058 cells were treated as described in C and nuclear lysates were immunoprecipitated with anti-total AKT or control IgG antibodies. Co-IP proteins were resolved on SDS-PAGE and immunoblotted for detection of p-AKT Ser⁴⁷³, total AKT and β-actin.