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. 2005 Jul;25(13):5329–5338. doi: 10.1128/MCB.25.13.5329-5338.2005

FIG. 7.

FIG. 7.

Regulation of MuSK promoter activity by endogenous CREB1. (A) CREB1-siRNA inhibited CREB1 expression. COS-7 cells were transfected with pKH3-CREB1 together with CREB1-siRNA or control siRNA (Con-siRNA) oligonucleotides. Expression of transfected CREB1 was revealed by immunoblotting with anti-HA antibody. (B) Effects of suppression of CREB1 expression on muscle differentiation. Transfected C2C12 myotubes were collected at different times after being switched to the differentiation medium and lysed. Lysates were probed for expression of MHC. (C) Increased MuSK promoter activity in C2C12 myotubes cotransfected with CREB1-siRNA. M715-Luc was cotransfected with pRL-CMV and pCMV-MyoD with CREB1-siRNA or Con-siRNA. Luciferase activities were assayed as in Fig. 2. Shown is relative luciferase activity (firefly/Renilla, mean ± the standard error of the mean) from a representative experiment done in duplicate and repeated three times with similar results. (D) Expression of MyoD99-170 reversed CREB1 inhibition of MyoD transactivation activity. M715-Luc was cotransfected with pRL-CMV and pCMV-MyoD with MyoD99-170. Luciferase activities were assayed as in Fig. 2. Shown is relative luciferase activity (firefly/Renilla, mean ± the standard error of the mean) from a representative experiment done in duplicate and repeated three times with similar results. (E). Expression of CREB1 inhibited dimerization of MyoD. Flag- or Myc-tagged MyoD was coexpressed without or with HA-CREB1 in COS-7 cells. Lysates were incubated with anti-Flag antibody and resulting lysates probed with anti-Myc antibody. IP, immunoprecipitation; IB, immunoblotting.