Fig. 3: Zinc induces CDK5 activation through Tyr15 phosphorylation. | Cell Death & Disease

Fig. 3: Zinc induces CDK5 activation through Tyr15 phosphorylation.

From: Zinc induces CDK5 activation and neuronal death through CDK5-Tyr15 phosphorylation in ischemic stroke

Fig. 3

a Zinc activated CDK5 in N2a cells. CDK5 activities were measured in cell lysates prepared from N2a cell incubated with or without ZnSO4 (10 μM) for 3 h by evaluating the phosphorylation level of Histone-H1. Representative blots with antibodies as indicated. b CDK5 activities were evaluated by calculating the ratio of phosphorylated to total Histone H1 levels in a (n = 3 cell experiment per group, t-test, p = 0.0009, ***p < 0.001). c Activation of CDK5 by zinc required the cellular signaling transduction. N2a cell lysates were incubated with or without ZnSO4 (10 μM) for 3 h, the direct effect of zinc on CDK5 activities were observed. d Quantitative analysis of CDK5 activities in c (n = 3 tube experiment per group, t-test, p = 0.35). e Zinc-induced CDK5 phosphorylation at Tyr15. Levels of total and Tyr15 phosphorylated CDK5, p35 and p25 in N2a cell lysates in a were detected by using the indicated antibodies. f Quantitative analysis of the protein levels in e (n = 3 cell experiment per group, t-test, from left to right, p = 0.0008, p = 0.0201, **p < 0.01, *p < 0.05). g Zinc inhibited calpain activity in N2a cells. Activities of calpain in the two groups were analyzed by ELISA assay (n = 3 cell experiment per group, t-test, p = 2.452E-05, ***p < 0.001). h GFP-tagged wild-type CDK5 (GFP-CDK5) or Y15F mutated CDK5 (GFP-CDK5-Y15F) were overexpressed in N2a cells with or without ZnSO4 (10 μM, 3 h) incubation. The expression of exogenous CDK5 and its phosphorylation at Tyr15 were evaluated by western blotting using the antibodies indicated. i Exogenously expressed CDK5 was immunoprecipitated with antibody against GFP and the CDK5 activities were detected by evaluating the phosphorylation level of Histone-H1. Representative blots with antibodies as indicated. j CDK5 activities were evaluated by calculating the ratio of phosphorylated to total Histone-H1 levels in i (n = 3 cell experiment per group, t-test, from left to right, p = 0.0051, p = 0.7020, **p < 0.01)

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