Primary myoblasts prepared from WT mice were treated with vehicle alone, 2 μM PD184352, 20 μM SP600125, or 20 μM SB230580 for 4h. (A) Relative mRNA levels of Pax7 mRNA. (B) Representative immunoblots from 2 independent experiments demonstrate the levels of PAX7 and phosphorylated and total c-JUN protein. (C) Densitometry quantification of PAX7 and phosphorylated and total c-JUN protein after treatment with various inhibitors. n = 4 in each group. (D) Relative mRNA levels of Pax7 and Jun after 72h of transfection of myoblasts with scrambled or c-JUN shRNA construct. (E) Relative mRNA levels of Pax7 and Jun in primary myoblasts after 72h of transfection with vector alone or c-JUN cDNA. (F) Relative mRNA levels of Pax7 in Traf6^–/– cultures upon transfection with c-JUN cDNA. n = 4 in each group. (G) Primary myoblasts were processed for ChIP assay for the binding of c-JUN at its putative consensus DNA sequence in Pax7 promoter. Agarose gel images of semi-quantitative RT-PCR demonstrate enrichment of Jun at indicated c-JUN/AP1 sites in mouse Pax7 promoter. The numbers indicate the position of consensus sequence upstream of first ATG in exon 1 of the Pax7 gene. (H) ChIP assay followed by qPCR analysis depicting percentage of input enrichment of c-JUN at specific sites in Pax7 promoter in Traf6^+/+ and Traf6^–/– cultures. n = 4 in each group. Error bars represent SD from mean. *P < 0.05 (vs. cultures treated with vehicle alone) by paired t test. ^§P < 0.05 (vs. corresponding cultures transfected with scrambled shRNA or vector alone) by unpaired t test. ^#P < 0.05 (vs. Traf6^+/+ cultures transfected with vector alone) by paired t test. ^‡P < 0.05 (vs. Traf6^–/– cells transfected with vector alone) by paired t test. ^†P < 0.05 (vs. Traf6^–/– cultures) by unpaired t test.