Research Paper Volume 4, Issue 7 pp 456—461

Figure 2. (a) ChIP assays were performed with EGR1(▭) and SIRT1 (▬) antibodies on non-stretched myotubes (ns), myotubes subjected to 30 min stretch and harvested immediately (s0) or 3 hours after (s3), immunoprecipitated DNA was analyzed by real time PCR with primers for the EGR1 binding sites and promoter occupancy was estimated as described in Methods. * and # indicates statistical significant difference from ns or s0, respectively. (b) C2C12 myoblasts grown on 24 well plates were transfected with the Sirt1 promoter reporter (0.1 μg/well) in the absence or presence of pcDNA-EGR1 and/or pcDNA-SIRT1 (0.2 μg/well); pcDNA was incorporated in the DNA mixtures to complete 0.5 μg/well and nicotinamide (NAM) to a 10 mM final concentration was added to the media when indicated. Luciferase activity was determined 24 hours after transfection with Dual Glo luciferase (Promega), * and # means statistical significant difference from empty vector or EGR1, respectively.