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Figure 2 | BMC Molecular Biology

Figure 2

From: Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment

Figure 2

SMAR1 binds to GAD65 promoter. A. Electro mobility Shift assay (EMSA) was done using GAD65 promoter DNA fragment which is bound by GST-SMAR1 in EMSA (lane 2) while cold competitor (C.C.; lane 3) reduced the binding showing the specificity of the interaction. B. Promoter fragments of GAD67 and actin was used as negative controls and did not show any binding with GST-SMAR1 proteins. C. Competitive EMSA shows SMAR1 interaction with GAD65 promoter (lane 3) is greatly reduced in presence of a competitor of cyclin D1 promoter oligo compared to no competition lane (lane 1). D. Supershift assay using Rin5f cell lysate without (lane 2) or with SMAR1 antibody (lane 3) showing shift in the GAD65 promoter band. The upper band in lane 3 represents GAD65-SMAR1-SMAR1 ab complex while lower band shows SMAR1-GAD65 promoter complex only. E. Luciferase assay showing that SMAR1 and p53 together leads to up-regulation of GAD65 promoter activity. Coexpression and knock down of SMAR1 and p53 in various combinations was used to look of luciferase activity of GAD65 promoter. F. Rin 5f cells were treated with FLAG-SMAR1 as well as siRNA of SMAR1 and western blot was carried out to test their effect over SMAR1 expression (upper panel). Similarly cells were treated with FLAG-p53 as well as siRNA of p53 and western blot using p53 antibody was done to check its expression levels (lower panel).

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