Figure 2

nPKCε isoform is located in the nerve terminal at the adult neuromuscular junction. (A1) shows a reconstruction of a NMJ stained for AChRs with fluorescent α-Bungarotoxin in red and immunolabeled with an antibody against nPKCε isoform in green. (A2-A4), several semiconsecutive confocal optical sections from the same NMJ shown in A1. The (*) indicates nPKCε-positive nerve terminal buttons accommodated in the AChRs delimited synaptic gutters. (B1) shows a reconstruction of a NMJ triple labeled for AChRs (fluorescent α-BTX in red), the Schwann cell (immunolabeled with an anti S-100 antibody in blue) and nPKCε isoform in green. (B2-B4), several confocal sections from the same NMJ shown in B1. The (*) also indicates nPKCε-positive granular immunolabeling concentrated on the nerve endings position over the synaptic gutters. The glia and muscle cell are not labeled. (C), Immunohistochemistry in semithin sections from a whole-mount multiple-immunofluorescent stained muscle. In the colocalization picture (C1), the triple staining that labeled nPKCε in green (C2), AChRs in red (C3) and Schwann cells in blue, (C4) shows the nPKCε immunolabel only in the middle of the sandwich formed by glia and muscle cell. In the inset (C1), the control triple staining that labeled PKCε (in green), AChRs (in red) and the nerve terminal (NT, in blue, neurofilament-200/syntaxin) shows that the nPKCε and nerve terminal markers are well colocalized. The bars in (A), (B) and (C) indicate 10 μm. The bar in the inset indicates 2.5 μm. The NMJ are from LAL muscles. (D) Western blot analysis of nPKCε, pnPKCε, cPKCα and syntaxin in denervated EDL muscle.