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. 2009 Jan 1;23(1):54–66. doi: 10.1101/gad.1717309

Figure 5.

Figure 5.

Myogenic differentiation of C2C12 cells is accompanied by an increase in the efficiency of SMD, a decrease in the efficiency of classical NMD, and an increase in the efficiency of alternative NMD. MBs or MTs (2 × 107) were transiently transfected with three plasmids: 3 μg of pmCMV-Gl Norm or pmCMV-Gl Ter, 12 μg of pFLUC-No SBS or pFLUC-SBS, and 3 μg of pRLUC. Two days later, cells were harvested. (A, left) Western blotting (WB) of myogenin, myoglobin, MYF-5, STAU1, UPF1, UPF2, UPF3X, BAG1, or c-JUN. Ponceau S staining was used as a loading control. (A, right) Quantitation of the level of STAU1, UPF2, or UPF3X relative to UPF1, or BAG1 or c-JUN relative to Ponceau S staining, in MBs and MTs. Each level in MBs was defined as 100. (B) Western blotting of specified proteins before (−) or after IP using anti-UPF1 or, to control for nonspecific IP, normal rabbit serum (NRS). (C, top and bottom) RT–PCR analysis of c-JUN or SERPINE1 mRNA after normalization to the level of GAPDH mRNA, where each normalized level in MBs was defined as 100. (D) The level of FLUC-No SBS or FLUC-SBS mRNA was normalized to the level of RLUC mRNA, and the normalized level of FLUC-No SBS in MBs or MTs was defined as 100. (E, top and bottom) RT–PCR analysis of BAG1 or TGM2 mRNA after normalization to the level of GAPDH mRNA, where each normalized level in MBs was defined as 100. (F) The level of PTC-free (Norm) or PTC-containing (Ter) Gl mRNA was normalized to the level of RLUC mRNA, and the normalized level of G1 Norm mRNA in MBs or MTs was defined as 100. (G, top and bottom) RT–PCR analysis of SC1.7 or SC1.6 mRNA after normalization to the level of GAPDH mRNA, where each normalized level in MBs was defined as 100 (asterisk denotes a nonspecific RT–PCR product that does not interfere with the analysis). (H, top and bottom) As in G. However, PAX3 mRNA or pre-mRNA was analyzed. (I, top and bottom) As in G. However, myogenin mRNA or pre-mRNA was analyzed. All results are representative of at least three independently performed experiments that did not vary by more than the amount shown.