Figure 1.

Immunolocalization of PKC isoenzymes and effect of BIM-1 on LPA-induced adducin phosphorylation in isolated guinea pig OHCs. (A) All PKC isoenzymes assayed were expressed in guinea pig OHCs (primary antibodies, green; DAPI, blue; rhodamine phalloidin, red). However, labeling was stronger for PKCα, PKCβ_I, and PKCε than for PKCβ_II, PKCδ, and PKCζ. In PKCβ_II and PKCδ, labeling was concentrated mostly at the subcuticular organ and the infranuclear region, whereas PKCζ immunoreactivity was frequently observed in the perinuclear region and below the cuticular plate (arrows). (B) LPA increased PKCα and PKCζ immunolabeling in the OHCs’ cytoplasm and nucleus. The effect on expression of PKCα, but not PKCζ, was prevented by pretreatment with BIM-1. LPA-induced increase in PKCα expression was associated with a similar increase in PKCα activation, as indicated by labeling with anti-p-PKCα at Ser-657, but only in the cytoplasm; no activation was observed in nuclear PKCα (primary antibodies, green; rhodamine phalloidin, red). (C) LPA also increased adducin phosphorylation of both Ser-726 and Thr-445 as indicated by reactivity to anti-p-adducin at Ser-726 and anti-p-adducin at Thr-445 antibodies (green; DAPI, blue; rhodamine phalloidin; red). However, treatment with BIM-1 reduced adducin phosphorylation only at Ser-726. In all images, scale bar = 10 μm.