Figure 5

Zinc Suppresses RANKL-induced Ca²⁺Oscillation in RAW264.7 cells without decreasing PLCγ1 activity. (A) RAW264.7 cells were cultured for 48 hours with RANKL (35 ng/ml) (n=3). Intracellular Ca²⁺ in single cells was measured using Fura-2/AM (5 μM). After observing RANKL-induced spontaneous Ca²⁺ oscillations for 10 minutes, ZnSO₄ (10 or 30 μM) was added to the bath solution. At the end of the experiment, ionomycin (5 μM) was added. We used Gd³⁺, a known calcium channel blocker, as a positive control. Data shown represent one experiment of three performed with similar results. (B) RAW264.7 cells were stimulated for 30 minutes with RANKL (35 ng/ml) or RANKL (35 ng/ml) plus ZnSO₄ (100 μM). Prepared proteins were analyzed by western blotting with anti-phospho-PLCγ1 or anti-PLCγ1 antibodies. Protein levels were quantified using densitometry.