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. 2012 Jun 6;32(23):8040–8052. doi: 10.1523/JNEUROSCI.4240-11.2012

Figure 1.

Figure 1.

Specificity of Munc13 isoform-specific antisera. A, Schematic representation of protein fragments (framed in green) used to raise specific antibodies against the canonical Munc13 isoforms/splice variants. C1, C1 domain; C2, C2 domain; MHD, Munc13 homology domain (Koch et al., 2000); MUN, Mun domain (Li et al., 2011). B–D, Western blot detection of Munc13-1, ubMunc13-2, bMunc13-2, and Munc13-3 using our own and commercial (panMunc13 TL; BD Biosciences) antibodies in homogenates from newborn brain (B), and adult retina (C, D) or olfactory bulb (D) homogenates of the indicated genotypes. The specificity of each antibody is indicated by the absence of corresponding immunoreactive band in extracts from the respective deletion-mutant mice. D, Immunoblot labeling with an anti-ubMunc13-2 serum revealed that ubMunc13-2 is strongly expressed in the olfactory bulb and that an unidentified protein is selectively expressed in Munc13-2-deficient but not in wild-type mice (right). A similar fragment was observed in Munc13-2-deficient retina extracts (left). E, In homogenates from Munc13-2 KO and Munc13-1/2 DKO hippocampal neurons, the ubMunc13-2 antibody recognizes a similar fragment. D, E, The asterisks indicate the 70–75 kDa fragments recognized by the ubMunc13-2 antibody in samples from Munc13-2-deficient mice. F, Single-plane confocal images taken upon immunocytochemical detection of Munc13 isoforms in vertical sections of adult mouse retina of the indicated genotypes. A weak, residual signal was observed for ubMunc13-2 at both the OPL and IPL of the Munc13-2 deletion-mutant retina. This may be consistent with the presence of a protein fragment observed in the Munc13-2 KO by Western blot (D). In contrast, the punctate labelings observed with the antisera directed against bMunc13-2 and Munc13-3 were no longer present in the corresponding KO retina. Note the occurrence of a nonspecific nuclear labeling obtained upon application of the antiserum directed against bMunc13-2. ONL, INL, Outer and inner nuclear layers; OPL, IPL, outer and inner plexiform layers; GCL, ganglion cell layer. Scale bars, 20 μm.