Figure 6.

Evidence that m½-sbsRNAs can influence C2C12 cell myogenesis by mediating SMD. (A–E) C2C12 MBs (5 × 10⁶ per 100-mm dish) were transiently transfected with 50 nM specified siRNA on day 0 and induced to differentiate to MTs by propagating in differentiation medium (DM) 1-d post-transfection. (A) Western blotting of the specified protein (see Supplemental Fig. 6A for siRNA-mediated down-regulation of each target at day 0), where the level of mCalnexin controls for variations in protein loading. Importantly, the same dilution standards in the left-most four lanes were used in all panels to control for variations in blotting and exposure times, and 10 μg of total cell protein was analyzed in the right-most seven lanes (see Supplemental Fig. 7A–G for blots showing these standards). (B) Plot of Western blotting of mTraf6 protein in the presence of control siRNA or ½-sbsRNA2(B2) siRNA as a function of days in DM, where the normalized level of each on day 0 is defined as 100% (see Supplemental Fig. 7A,C,F, H for data). Dashed lines denote time points at which ½-sbsRNA2(B2) siRNA no longer down-regulated its target. (C) As in B but showing RT–PCR quantitations of mTraf6 mRNA/mTraf6 pre-mRNA (see Supplemental Fig. 7H for data). The dashed lines are as in B. (D) As in B except mCdc6 protein was analyzed (see Supplemental Fig. 7A,D,G,I for data). Dashed lines denote time points at which ½-sbsRNA3(B4) siRNA no longer down-regulated its target. (E) As in C but showing mCdc6 mRNA/mCdc6 pre-mRNA (see Supplemental Fig. 7I for data). The dashed lines are as in D. All results are representative of at least two independently performed experiments that did not vary by more than the amount shown.