Figure 1. PKC-δ activation during cisplatin treatment in mice and RPTCs.

(A) Kinase assay of PKCδ activity in kidney tissues. Male C57BL/6 mice of 8 to 10 weeks of age were injected with 30 mg/kg cisplatin before collection of renal tissues at day 0–3. PKCδ was immunoprecipitated from tissue lysate and in vitro kinase reaction with the substrate histone H1 and [γ-32P]ATP. Histone H1 phosphorylation was analyzed by SDS-PAGE and autoradiography to indicate kinase activity. (B) PKCδ phosphorylation at tyr-311 in kidney tissues. Kidney tissue lysate was analyzed by immunoblot analysis for phosphorylated (tyr-311) PKCδ (p-PKCδ), total PKCδ, or β-actin. (C) Tyrosine phosphorylation of PKCδ during cisplatin treatment in vivo. PKCδ was immunoprecipitated from control and cisplatin-treated renal tissues for immunoblot analysis of phosphotyrosine (pY). (D) PKCδ (tyr-311) phosphorylation during cisplatin treatment in vitro. RPTCs were treated with 20 μM cisplatin for 0 to 4 hours to collect whole cell lysates for immunoblot analysis of total and phosphorylated (tyr-311) PKCδ. (E) In vitro kinase assay of PKCδ activation in RPTCs. RPTCs were treated with 20 μM cisplatin for 0 to 16 hours to collect whole cell lysates for PKCδ immunoprecipitation and kinase activity assay. (F) Translocation of PKCδ during cisplatin treatment. RPTCs were treated with cisplatin for 0 to 1 hours and then fractionated into nuclear, membrane, and cytosolic fractions for immunoblot analysis of PKCδ. Mean ± SD, n = 4. *P < 0.001 versus control.