Fig 2: Munc18-1 expression at the nerve terminal is regulated by PKC activity. (A) Representative confocal doubly-immunofluorescence-stained NMJs images (Munc18-1 and AChR) after a treatment with PBS as a control, PMA, and eV1-2 peptide. PMA increases Munc18-1 fluorescence intensity while eV1-2 peptide decreases it. Scale bar = 10 µm. (B) Fluorescence intensity quantification of Munc18-1. Data are mean value ± SEM, *p < 0.05.

Fig 3: nPKCe and cPKCßI modulate Munc18-1 phosphorylation under muscle contraction conditions. (A) Representative Western blot bands and quantification show that Munc18-1 phosphorylation and the pMunc18-1/Munc18-1 ratio are significantly decrease after stimulation resulting in contraction in nPKCe inhibitor peptide eV1-2 preincubated muscles. (B) Representative Western blot bands and quantification show that the pMunc18-1/Munc18-1 ratio are significantly decreases after stimulation resulting in contraction in cPKCßI inhibitor peptide ßIV5-3 preincubated muscles. (C) Representative Western blot bands and quantification show that the phosphorylation of nPKCe and the pnPKCe/nPKCe ratio significantly increases after stimulation resulting in contraction in cPKCßI inhibitor peptide ßIV5-3 preincubated muscles. Data are mean percentage ± SEM, *p < 0.05 (n = 5).

Fig 4: Munc18-1 is exclusively located in the presynaptic component of the NMJ. Multiple-immunofluorescence-stained muscles visualized at the confocal microscope. (A) Munc18-1 colocalizes with AChR from a NMJ en face view. (B) Munc18-1 colocalizes with AChR from a NMJ en side view. (C,D) Munc18-1 colocalizes with syntaxin and AChR from en face view. (E) Munc18-1 colocalizes with syntaxin and AChR from en side view. (F) Munc18-1 colocalizes with S-100 and AChR from en face view. (G) Munc18-1 colocalizes with S-100 and AChR from a NMJ en side view. Scale bars = 10 µm.

Fig 5: BDNF/TrkB signaling modulates Munc18-1, pMunc18-1, nPKCe, and pnPKCe levels under synaptic activity conditions. (A) Representative Western blot bands and quantification show that after the treatment with BDNF, the ratio of pMunc18-1/Munc18-1 significantly decreases while after the 47/TrkB treatment, it significantly increases. (B) Representative Western blot bands and quantification show that after the 47/TrkB treatment, pnPKCe significantly increases without affecting nPKCe levels. In accordance, the ratio is significantly increased. Data are mean percentage ± SEM, *p < 0.05 (n = 5).