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. 2008 Dec 31;40(6):596–606. doi: 10.3858/emm.2008.40.6.596

Figure 2.

Figure 2

Essential role of PKCµ in hypertonicity-induced HSP70 expression. (A) Phosphorylation status of various PKC isoforms under hypertonic conditions. Cells were maintained in media containing an additional 130 mM NaCl for the indicated time periods. Cell lysates were blotted with anti-p-PKCα/βII, anti-p-PKCζ/λ, anti-p-PKCθ, anti-p-PKCδ, and anti-p-PKCµ antibodies. (B) Effect of PKCµ inhibitor, Gö6976 on hypertonicity-induced HSP70 expression. Cells were pretreated with Gö6976 (5 µM or 10 µM) for 1 h, and then kept in hypertonic media for either 30 min for the detection of p-PKCµ or 12 h for the detection of HSP70. Cell lysates were resolved via SDS-PAGE and blotted with anti-p-PKCµ and anti-PKCµ antibodies (upper panel) or anti-HSP70 antibody (low panel). (C) Effect of kinase-inactive mutants (KR) of PKCα and PKCβI on hypertoniciy-induced HSP70 expression. Cells were transiently transfected with HA-PKCα KR or HA-PKCβI KR constructs. After maintaining transfectant cells in hypertonic media for 12 h, the cell lysates were prepared. Exogenous PKCα and PKCβI (indicated by arrow) and HSP70 were detected with anti-HA antibody. The asterisk designates non-specific signals. (D) Effect of PKCµ inhibitor on HSP70 expression in hypertonically stressed M-1 cells. Human renal epithelial M-1 cells were pretreated with inhibitors (25 µM GF109203X or 10µmM Gö6976) for 1 h and exposed to hypertonicity for the indicated time periods (upper panel) or 12 h (lower panel). Cell lysates were resolved on SDS-PAGE and blotted with anti-HSP70 and anti-p-PKCµ antibodies. (E) Effect of PKCµ knock-down on hypertonicity-induced HSP70 expression. NIH-3T3 cells were transiently transfected with RNAi duplexes (25 µM negative control or 25 µM PKCµ RNAi duplexes). After 48 h of transfection, the cells were exposed to hypertonic conditions for 12 h. The cell lysates were resolved via SDS-PAGE and blotted with anti-HSP70 and anti-PKCµ antibodies. (F) Effect of PKCµ knock-down on hypertonicity-induced HSP70 mRNA transcription. Cells were transiently transfected with PKCµ RNAi duplexes. After 48 h of transfection, the cells were exposed to hypertonic conditions for 4 h. Cells were harvested and real time PCR was conducted as described in Materials and Methods.