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. 2009 Jul 22;284(39):26502–26509. doi: 10.1074/jbc.M109.018150

FIGURE 1.

FIGURE 1.

Expression and subcellular localization of P1c in the central nervous system. A, expression of P1c during brain development assessed by immunoblotting of brain homogenates from mice of different ages. Antibodies (Abs) used are indicated on the left. MAP1B HC, MAP1B heavy chain; E15, embryonal stage day 15; P0, P7, and P14, 0, 7, and 14 days after birth; Pw12, Pw52, and 1c−/−-Pw12, 12- and 52-week-old WT and 12-week-old P1c−/− mice, respectively. Tubulin served as loading control. Note that (i) A1c Abs label bands corresponding to full-length P1c (∼520 kDa) and a rodless P1c variant (∼390 kDa); (ii) pan-plectin (N-terminal) antibodies label bands corresponding to all full-length (∼520 kDa) and rodless (∼390 kDa) variants irrespective of their alternative first cooling exons (see also B); (iii) in adult mice (Pw12 and Pw52), rodless P1c is detectable only at low levels; and (iv) contrary to P1c, MAP1B is expressed at early developmental stages. B, schematic diagram of P1c protein variants. Boxes represent main domains of P1c. Gray boxes, amino acid sequence encoded by the alternative first exon 1c; N domain, N-terminal globular domain; rod, central coiled-coil rod domain; C domain, C-terminal globular domain of P1c. Exons coding for these domains are indicated. Alternative splicing events of different first exons into exon 2 are depicted by a straight line, the splice event leading to excision of exon 31 is indicated with a kinked line. The estimated molecular masses of full-length and rodless plectin variants are indicated. Note that A1c exclusively recognizes the exon 1c-encoded domain, whereas pan-plectin (N-terminal) recognizes epitopes in the N-terminal globular domain which is common to all plectin isoforms known so far. C–F, IFM of cortex sections from WT and P1c−/− animals using primary Abs as indicated. Note, Abs A1c and pan-plectin (number 46) both reveal punctuate staining patterns in WT but not P1c−/− cortex, establishing P1c as the dominating isoform in the cortex. Nuclei are stained with Hoechst dye. G–J, and L, IFM of fourth ventricle ependyma from WT and P1c−/− animals using primary Abs as indicated. Note (i) the residual staining of knock-out ependyma with anti-pan-plectin antiserum in I (indicating that isoforms other than P1c are additionally expressed in these cells) and (ii) P1c expression in ependymal cells and underlying astrocytes in L. K and M–O, IFM of WT cortex sections. Note that (i) punctuate P1c staining in K does not overlap with GFAP-positive astrocytes; (ii) the partial colocalization of P1c and MAP2 observed in longitudinally cut dendrites (M) is barely observed in cross-cut dendrites (N), where P1c- and MAP2-specific signals nevertheless are found in close vicinity to each other; and (iii) P1c is not localized in anti-synaptophysin (Synap)-immunostained presynaptic vesicles (O). P and Q, IEM of P1c-specific diaminobenzidine reaction product in WT (P) and P1c−/− (Q) cortex. Note the specific labeling of non-myelinated postsynaptic dendrites. Arrowhead, presynaptic vesicles. Scale bars, 25 μm (G–J and L), 20 μm (C–F, K, and O), 5 μm (M and N), and 0.3 μm (P and Q).