Fig 2: Assessing the interaction of ELF1 or MAZ with DNA. (a, b) Electrophoretic mobility Shift Assay (EMSA). 40 fmol of 5' biotin-conjugated doubled stranded DNA fragment (shown is the upper strand) containing the wild type (red font) or mutant (underlined) ELF1 or MAZ response element (RE) derived from the CXXC5 core promoter was incubated with nuclear extracts (NE, 45 µg) of MCF7 cells in the absence or presence (+) of an antibody (Ab) or 250-fold excess unbiotinylated DNA (UnB) with wild type RE. Free indicates the unbound biotinylated RE. Asterisks (red) indicate protein-bound DNA. (c) MCF7 cells were transfected with pGL3 bearing none (B), the wild-type (SegA), or Segment A with a deletion of ELF1-RE (SegA?ELF1-RE), or MAZ-RE (SegA?MAZ-RE) as promoter driving Luciferase cDNA expression as the reporter without or with an expression vector bearing the HA-ELF1 or HA-MAZ cDNA for 24 h. The transfection efficiency was monitored by the co-expression of pCMV-Renilla Luciferase. Cellular extracts were then subjected to dual-luciferase assays. Asterisks (*) indicate significant differences depicted as fold change compared to B which was set to 1; whereas the superscript “a” denotes significant differences compared to SegA. (d, e) MCF7 cells were transfected with an expression vector bearing none (EV), HA-tagged ELF1, or HA-tagged MAZ cDNA. Isolated RNA was converted into cDNA libraries and followed by qPCR using either CXXC5-specific or OAS1-specific for ELF1 control or MYC-specific for MAZ control primers. Results were normalized with the RPLP0 expression using the 2-??CT method. (f, g) Nuclear extracts of untransfected (UT) or transiently transfected MCF7 cells with control siRNA (CtS), a pool of siRNA specific (siR) to ELF1 (f) or MAZ (g), or with an expression vector bearing the HA-ELF1 or the HA-MAZ cDNA for 48 h were subjected to IB using ELF1 or MAZ antibody. Membranes were then re-probed with the HA antibody. Molecular masses (MM) in kDa are indicated. (h, i) Total RNA from MCF7 cells transiently transfected cells with control siRNA (CtS), siRNA specific (siR) to ELF1 (h), or MAZ (i) were processed for and subjected to qPCR using primers specific to CXXC5, the OAS1, or MYC. Asterisks (*) denote significant differences depicted as fold change compared to CtS of three independent experiments.