Figure 5. RelA/p65 promotes Rapsyn transcription. A, Schematic diagram showing Rapsyn reporter construct R5k-Luc that contains the 5′-flanking region of Rapsyn gene and the downstream cDNA encoding firefly luciferase. Four putative NF-κB binding sites (A–D) were indicated in the R5K promoter (A, −3510 to −3500 nt; B, −2991 to −2981 nt; C, −2208 to −2199 nt; D, −593 to −583 nt). B, C2C12 myotubes were cotransfected with R5k-luc and GFP-p65, MyoD, or control plasmid. Firefly and Renilla luciferase activity was assayed as described in Materials and Methods. *p < 0.05, **p < 0.01, Student's t test. C, R5K-Luc (marked as ABCD) or the constructs with the deletion of indicted NF-κB binding sites (mutations lacking site A, C, D, AC, or AD were labeled as BCD, ABD, ABC, BD, or BC, respectively) were cotransfected with GFP-p65 or vehicle plasmid. The data shown are the ratio of relative reporter activity between p65-transfected and control cells. All the experiments were repeated at least six times. *p < 0.05, one-way ANOVA with Turkey's honestly significant difference post hoc tests. D, Nuclear extracts were prepared from C2C12 myotubes, and EMSA was performed with biotin-labeled probes corresponding to putative NF-κB binding sites. The MHC-I probe containing a bona fide NF-κB binding site was taken as a positive control and the mutated NF-κB binding site was taken as negative control (N.C.). E, Supershift EMSA was performed with myotube nuclear extracts preincubated with normal IgG or specific antibodies against RelA/p65 or p50 subunits. F, Nuclear extracts from C2C12 myotubes were incubated with normal IgG or antibodies against RelA/p65, p50, or acetylated histone H3; quantitative PCR analysis was conducted with coimmunoprecipitated DNA using Rapsyn promoter primers covering the region containing site D. Error bars indicate SEM.