Fig. 3

Pamidronate restores the altered phosphorylation of AKT, ERK1/2, and NF-κB p65 in bilirubin-impaired SHED. SHED were cultured as described in Fig. 1a. a–c Sequential expression of AKT, ERK1/2, and NF-κB p65 and their phosphoproteins, p-AKT (Ser), p-AKT (Thr), p-ERK1/2, and p-NF-κB p65, was analyzed at the indicated time by western blot analysis. Representative images of western blotting were shown. d SHED were stimulated with 0 μM bilirubin (B0), 50 μM bilirubin (B50), and 50 μM bilirubin plus 10 μM pamidronate (B50+PAM) under serum-depleted condition for 2 h. Nuclear translocation of NF-κB p65 in SHED by immunofluorescent microscopy. Nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images of the localization of NF-κB p65 in SHED after 2 h of the stimulation. Bars = 20 μm. a–c n = 5 for all groups. Statistical analysis was performed as described in the “Methods” section. Graph bars showed the means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.005 (vs. 0 h in each group). NS, no significance