Figure 4.

Zinc Inhibits RANKL-induced Nfatc1 Activation by suppressing NFATc1 Translocation to the Nucleus in RAW264.7 cells. (A, B) RAW264.7 cells were incubated with RANKL (35 ng/ml) alone or RANKL (35 ng/ml) with various concentrations of ZnSO₄. After 30 minutes, cytosolic and nuclear fractions were extracted from each group and evaluated by western blotting with the anti-phospho-Nfatc1 antibody (A, upper panel and C, left panel), anti-Nfatc1 antibody (A, lower panel, C, right panel), or anti-PP2B-Aα antibody (B), which is the catalytic subunit of calcineurin. Subcellular fraction purity and equal sample loading were evaluated by analyzing Lamin B and α-tubulin. Protein levels were quantified using densitometry. (C) RAW264.7 cells were incubated for 30 minutes with RANKL (35 ng/ml), RANKL (35 ng/ml) with ZnSO₄ (100 μM), or RANKL (35 ng/ml) with FK506 (1 μM). Cytosolic phospho-Nfatc1 and nuclear Nfatc1 were analyzed using western blot. (D, E) RAW264.7 cells were stimulated with RANKL (R) or RANKL (R) plus ZnSO₄ (30 or 100 μM) for 30 minutes. Nuclear fractions were prepared, and the transcriptional and DNA binding activity of Nfatc1 and NF-κB were measured using luciferase reporter assay and ELISA, respectively. RLU, Relative Light Units (F) RAW264.7 cells were cultured as shown in panel C. Cytosolic PP2B-Aα was examined by western blot and calcineurin activity was compared with the treated groups. Data are presented as the mean ± S.D. of three independent experiments; * p < 0.05 compared to RANKL (R).