mmlv clotech ca and rnasin promega wi cdna was amplified by real time pcr using the opticon ii mj research the dynamo sybr green 1 kit finnzyme finland was used according to the manufacturer 39 s instructions primers were specifically designed between two adjacent exons using the autoprime program either custom made or commercial inventoried taqman probes from applied biosystems applied biosystems ca were also used according to the manufacturer 39 s instructions mrna levels for each gene were normalized to those of hprt using the u03b4 u03b4ct method primer and probe sequences are available upon request cdk9 42 protein levels are not affected during the differentiation program while its kinase activity is clearly augmented and strictly required for myod mediated muscle specific transcription and myotube formation simone et al oncogene 21 4137 4148 2002 giacinti et al j cell physiol 206 807 813 2006 simone and giordano cell death differ 14 192 195 erratum in cell death differ 14 196 2007 cdk9 55 is synthesized upon the induction of muscle differentiation c2c12 cells either undifferentiated gm or induced to differentiate dm for the indicated times were analyzed using different techniques see fig1 a 1e c2c12 mouse myoblasts were cultured and stimulated to terminally differentiate into myotubes the cdk9 55 isoform was significantly upregulated in cells induced to differentiate while cdk9 42 displayed similar levels between proliferating and differentiating cells fig1 a furthermore cdk9 55 expression preceded myogenin and mhc expression fig1 a nuclear extracts were prepared as described in de falco et al oncogene 21 7464 7470 2002 cdk9 55 localized into the nucleus and its upregulation coincided with the hyperphosphorylation of the ctd of rnapolii fig1 b the induction.