Research Paper Volume 12, Issue 8 pp 7042—7055

Figure 5. CREB is involved in the regulation of OGT expression. HEK-293A cells were transfected with pCI-neo, pCI/HA-CREB or siRNA of CREB respectively. (A) mRNA levels of OGT and GAPDH were measured by RT-PCR. The quantification of relative mRNA level of OGT after normalization with the mRNA level of GAPDH was represented as mean ± S.D. (n = 3); *, p < 0.05, **, p < 0.01. (B) Protein levels of OGT, CREB and GAPDH were examined by western blot using anti-OGT, anti-CREB and anti-GAPDH antibody. Relative OGT or CREB level was quantified after normalization with the protein level of GAPDH and presented as mean ± S.D. (n=3), *, p < 0.05; **, p < 0.01. (C) pCI/HA-CREB was co-transfected with pGL3/OGT₁₅₀₀ into HEK-293T cells. HA-CREB was immunoprecipitated with anti-HA antibody. Co-immunoprecipitated DNA of OGT promoter with CREB was determined by PCR with three sets of primers specific to CRE elements as indicated for amplifying the DNA. The PCR product was separated by agarose electrophoresis. (D) In HEK-293A cells transfected with pGL3/ OGT₁₅₀₀ and pRL-TK, pCI-neo, pCI/HA-CREB or siRNA of CREB was transfected with or without overexpression of SIRT1. After 48 h transfection, the luciferase activity was measured and normalized with Renilla luciferase. The relative activity of luciferase was presented as mean ± S.D. (n=3), *, p < 0.05, **, p < 0.01, ***, p< 0.001 versus control group; ###, p< 0.001. (E) pCI-neo, pCI/HA-CREB or siRNA of CREB was transfected into HEK-293A cells overexpressing SIRT1 or not. Protein levels of SIRT1, OGT, CREB or GAPDH were detected by western blot using anti-Myc, anti-OGT, anti-CREB or anti-GAPDH antibody. Relative OGT or CREB level was quantified after normalization with the protein level of GAPDH and presented as mean ± S.D. (n=3), *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control group; ##, p< 0.01.