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. 2013 Jan 28;288(12):8585–8595. doi: 10.1074/jbc.M112.413997

FIGURE 3.

FIGURE 3.

A, DPP activates CaMKII in C3H10T1/2 cells. C3H10T1/2 cells were stimulated with rDPP (500 ng/ml), and Western blotting was performed. Experiments were done in triplicate. *, #, and [caret], p < 0.05 as compared with untreated cells. B, DPP activates CaMKII in HMSCs. C3H10T1/2 cells were stimulated with rDPP (500 ng/ml), and Western blotting was performed with Abs against CaMKII. Experiments were done in triplicate. *, #, and [caret], p < 0.05 as compared with untreated cells. C, DPP-mediated activation of CaMKII can be inhibited by the treatment with BAPTA-AM. C3H10T1/2 cells were pretreated with BAPTA-AM (50 μm) followed by stimulation with rDPP for the indicated time points, and Western blotting was performed with Abs against CaMKII. Experiments were done in triplicate. *, #, and [caret], p < 0.05 as compared with untreated cells. D, CaMKII inhibitor KN-62 inhibits DPP-induced [Ca2+]i release in C3H10T1/2 cells. DPP-induced [Ca2+]i was measured in confluent C3H10T1/2 cells loaded with 10 μm KN-62 for 30 min prior to the addition of Fura-2AM. Fura-2AM-loaded cells were washed three times, placed in Ca2+- and Mg2+-free HBSS, and then stimulated with rDPP (500 ng/ml). The experiment was repeated four times, and a representative plot is shown. Picture images display DPP-induced [Ca2+]i release in KN-62-pretreated C3H10T1/2 cells at 250 s. E, KN-62 inhibits CaMKII activation in C3H10T1/2 cells. Treatment of C3H10T1/2 cells with KN-62 (10 μm) inhibits CaMKII activation. Total proteins were isolated, and Western blots were developed with CaMKII antibody and anti-phospho-CaMKII antibody. Equal protein loading was confirmed by stripping the blot followed by probing it with tubulin antibody. Experiments were done in triplicate. *, #, and [caret], p < 0.05 as compared with untreated cells. F, KN-62 inhibits DPP-mediated phosphorylation of CaMKII in HMSCs. Total proteins were isolated from DPP-stimulated HMSCs, and immunoblots were performed with CaMKII antibody and anti-phospho-CaMKII antibody. Experiments were done in triplicate. *, #, and [caret], p < 0.05 as compared with untreated cells. C, control.