Research Paper Volume 11, Issue 10 pp 3094—3116

Tyrosine nitrations impaired intracellular trafficking of FSHR to the cell surface and FSH-induced Akt-FoxO3a signaling in human granulosa cells

Figure 4. PN attenuated Akt-FoxO3a signaling. (A) PN attenuated Akt-FoxO3a activation and nuclear export of FoxO3a. KGN cells were incubated with or without PN (100 nM, 12 hrs), SC3036 (30 μM, 4 hrs) and LY294002 (30 μM, 4 hrs) followed by treatment with FSH (1 nM, 4 hrs). Relative protein expressions of p-Akt, Akt, p-FoxO3a, FoxO3a in cytoplasmic fractions (β-actin as internal standard) and FoxO3a in nuclear fractions (Lamin B1 as internal standard) were determined by immunoblots. (B) PN strengthened FoxO3a binding-dependent luciferase activities. KGN cells were transfected with the pGL3-Foxo3a consensus binding element-luciferase plasmids, and were incubated with or without PN (100 nM, 12 hrs), SC3036 (30 μM, 4 hrs) and LY294002 (30 μM, 4 hrs) followed by treatment with FSH (1 nM, 4 hrs). The luciferase activities were determined using the Dual-Luciferase Reporter Assay System. Open triangle: p<0.05 vs. Group 1; Bold triangle: p<0.05 vs. Group 2 (n = 3–6).