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. 2013 Jan 22;41(5):3240–3256. doi: 10.1093/nar/gks1227

Figure 2.

Figure 2.

CREB binds to the CRE-like elements in human GLUT3 promoter and promotes the luciferase expression driven by this promoter. (A) Schematic diagram of three CRE-like elements in GLUT3 promoter and its luciferase reporter. (B) CREB bound to three CRE-like elements detected by EMSA. 32P-labeled consensus CRE (Con) as a positive control and three CRE-like elements, CRE1, CRE2 and CRE3, of human GLUT3 promoter were incubated with the nuclear extracts (5 µg for consensus CRE and 10 µg for three CRE-like elements) in the absence or presence of anti-CREB or anti-P-CREB and subjected to native PAGE. The gel was dried and autoradiographed with a PhosphorImager (BAS-1500, Fijifilm, Japan). (C) CRE-like elements in human GLUT3 promoter were co-immunoprecipitated with CREB by ChIP assay. SH-SY5Y cells under differentiation condition were transfected with pCI/CREB and then CREB was immunoprecipitated by anti-HA. Three CRE-like elements in the immune-complexes were amplified by PCR using the primers specific to CRE1, CRE2 or CRE3. The PCR products were quantified by densitometry and presented as mean ± SD (n = 3); **P < 0.01. (D) Forskolin treatment enhanced the binding of CREB with the CRE of GLUT3 promoter. SH-SY5Y cells under differentiation condition were transfected with pCI/CREB, treated with forskolin for 10 h and then CREB was immunoprecipitated by anti-HA. The three CREs were amplified by PCR with their specific primers. (E) Promoter of human GLUT3 promoted luciferase expression. Human GLUT3 promoter (−2000 to +241) was inserted into pGL3-basic vector to generate pGL3/GLUT3−2000. pGL3/GLUT3−2000 or its control pGL3 showed in panel A was co-transfected with pRL-TK into HEK-293T cells for 48 h. The firefly luciferase activity and Renilla luciferase activity were measured subsequently and the firefly luciferase activity was normalized with Renilla luciferase activity and presented as mean ± SD (n = 3); **P < 0.01. (F) CREB enhanced GLUT3 promoter-driving luciferase expression. pGL3/GLUT3−2000 and pRL-TK was co-transfected with pCI (Con) or pCI/CREB into HEK-293T cells for 48 h. The luciferase activity was measured and presented as mean ± SD (n = 3); **P < 0.01.