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Fig. 1. Characterization of MURF-2 expression patterns in striated muscle. (A) MURF-2 contains a RING-finger domain (green), the MURF family conserved region (red), a B-box domain (yellow), two coiled-coil domains (blue), and an acidic tail (orange). Previous mRNA studies predicted four potential MURF-2 isoforms generated by differential splicing events within the 3'-region: p60 (full-length, lower schematic), p50 (upper schematic), a p27 isoform (missing the N-terminal region; not shown) and p60B (containing an alternate C-terminus generated by translation of alternative frames in the terminal exon; not shown). Two different antibodies were generated against the C-terminal region of mouse MURF-2. Antibodies against a synthetic peptide (the extreme 17 C-terminal residues) are predicted to recognize the full-length p60 MURF-2, as well as the p50 and p27 forms. Antibodies also were generated against an internal 83-residue, recombinant fragment containing the acidic tail domain (residues 278-365) shared by the p60, the p50, and the potential p60B splice forms. For further description of the potential p27 and p60B isoforms (see Pizon et al., 2002). Positions corresponding to the antisense oligonucleotides (B, C, and D) for chick MURF-2 also are indicated. (B) Anti-C-terminal MURF-2 antibodies recognize a broad band (55-60 kDa) in fetal rat heart lysates (lane 1) and two distinct bands (~55 and ~60 kDa) in adult rat heart lysates (lane 2), in primary cultures of embryonic chick cardiac myocytes (lane 3), and in primary cultures of chick skeletal myocytes (lane 4: myoblasts; lane 5: myotubes at day 3 of culture). (C) Immunofluorescence staining revealed that MURF-2 is detected diffusely in the cytoplasm, and at the M-line region of a portion of myofibrils (a: MURF-2, red; b: {alpha}-actinin, green; merged inset in b; double arrows point to striations). In the same culture, MURF-2 also was detected in a discontinuous, spotted pattern along a portion of microtubules in many chick cardiac myocytes (c: MURF-2, red; d: {alpha}-tubulin, green; merged inset in d; arrow points to tubulin staining). MURF-2 was not detected in fibroblasts (data not shown). Bar, 10 µm.