Figure 5. UFD2a-7 and UFD2a-7/7a mRNA and protein expression are upregulated during differentiation and regeneration.

A). C2C12 cells were plated in growth medium overnight and then transferred into differentiation medium (DM). Cell cultures were harvested for analysis after the indicated times. Equal total protein was loaded on SDS-PAGE (see Figure S1 for poneau S staining) and analyzed for expression of the indicated proteins. The UFD2a panel was performed on a large-format 6% bis-acrylamide gel for greater separation of the 2 larger UFD2a isoforms and probed with the 15041 polyclonal antibody, which recognizes all isoforms. B). C2C12 cells were plated in growth medium overnight and then transferred into DM. Cell cultures were harvested for analysis after the indicated times. Total RNA was isolated using TRIzol and used as a template for RT-PCR. PCR was performed on the resulting cDNA (made with or without reverse transcriptase (RT)) using a 5′ primer complementary to exon 6 and a 3′ primer specific for either the exon 7/exon 8 junction (top panel) to detect UFD2a-7 transcripts or the exon 7a/exon 8 junction (middle panel) to detect UFD2a-7/7a transcripts. Primers specific to exon 19 and 20 were used in the bottom panel to show total UFD2a transcripts. C). Total RNA was isolated with TRIzol from duplicate samples of cells used in panel A and subjected to RT-PCR. Quantitative PCR was performed in duplicate using TaqMan primers that recognized the indicated transcripts. Transcript expression data, normalized to the expression of HPRT, are expressed relative to the expression level at time zero. Error bars indicate standard deviation.