Figure 4. B55α knockdown inhibited oxidative stress-induced FOXO1 dephosphorylation and nuclear translocation.

(A) INS-1 cells were co-transfected with GFP–FOXO1 and B55α siRNA or scrambled siRNA, and immunoblotted for GFP–FOXO1 and endogenous FOXO1 using antibodies against phosphorylated (p) Thr²⁴ and Ser²⁵⁶ and total FOXO1, as well as against B55α. Cells were incubated in the presence (+) or absence (−) of 100 μM H₂O₂ for 1 h. GFP–FOXO1 (97 kDa) is distinguished from endogenous FOXO1 (70 kDa) by its slower migration. (B) Ratios of phospho-Thr²⁴ and phospho-Ser²⁵⁶ phosphorylated FOXO1 relative to total FOXO1 in untreated (−) or H₂O₂-treated (+) cells transfected with scrambled or B55α siRNA. Ratios were normalized to untreated cells transfected with scrambled siRNA. Band intensities were acquired from (A) using Bio-Rad quantity analysis software and represent the means±S.D. of four samples. (C) INS-1 cells were co-transfected with GFP–FOXO1 and B55α siRNA or scrambled siRNA. GFP localization was visualized by fluorescence microscopy in untreated and H₂O₂-treated cells. (D) Percentages of GFP-positive nuclei after H₂O₂ treatment in scrambled siRNA or B55α siRNA-transfected cells. At least 150 cells were counted in three independent experiments. *P <0.01.