Five days after CTX injection, UFD2a-7 and UFD2a-7/7a BMS-907351 expression returned, and by 12 days, UFD2a-7 was almost undetectable, suggesting that UFD2a-7 is in fact a transient alternative splice form that may be important during the differentiation process. This switch in UFD2a isoform expression mirrored that of MHC, a well-known marker of mature muscle tissue. The transcription factor Myf5, which is expressed only in proliferating myoblasts, was not detected prior to injection, was induced at 2 to 5 days postinjection, and then returned to undetectable levels within 12 days, presumably upon completion of regeneration. These studies provide a compelling link between UFD2a isoform expression and the carefully regulated process of muscle regeneration. Since the molecular mechanisms that regulate adult myoblast differentiation are Perifosine Akt inhibitor thought to be similar to those that regulate embryonic myogenesis, we analyzed UFD2a isoform expression during development in murine heart and skeletal muscle. At all developmental time points tested, significantly more ubiquitous UFD2a isoform was present than was found in adult muscle tissue, which most likely indicates that myoblasts and other contaminating cells types were actively dividing as the tissues continued to grow and develop. While the expression patterns discussed above were robust in western blots performed using samples from multiple mice, It was noted that there was some variability in the relative levels of each isoform detected at individual developmental time points. Therefore, a second set of skeletal muscle and heart tissue samples is shown in Figure S2. To examine whether the sequential transition from Ufd2a-7 to UFD2a-7/7a during development is conserved, we examined the pattern of ube4b mRNA expression during early development in zebrafish by using RT-PCR. We specifically examined the mRNA expression pattern of ube4b, rather than protein levels, because neither rabbit polyclonal nor mouse monoclonal antibodies specifically recognized zebrafish Ube4b by Western blotting. Nested PCR performed on the purified products obtained from 10-, 12-, 24-, and 48-hpf embryos and 7-dpf embryos using primers F1 and R1 followed by F2 and R2 revealed that more of Ufd2a-7 was present at the earlier developmental stages. These 2 nested PCR products were sequenced and blasted against the NCBI zebrafish ube4b genome. An identical match was found with 2 sequences within what had been previously considered intronic sequence in the zebrafish UFD2a ortholog. These results confirmed that the smaller amplicon was consistent with Ube4b-7, and the larger amplicon seen in increasing amounts from 12 hpf to 7 dpf represented the larger Ube4b-7/7a isoform. We have entered the two novel Ube4b isoforms in Genebank and the accession numbers: JF289275 and JF289276 have been assigned.