Fig. 6
Shift assays of EGR1 binding to probe [−71 to −98] derived from the αAtp2b2 transcript promoter. All blots shown are 10 min exposures. a Western blot of EGR1 protein expression in NIH/3T3 or HeLa cells. Nuclear and cytoplasmic fractions were compared. HeLa nuclear extract was used for shift assays due to its detectable expression of EGR1. b The [−71 to −98] probe was used in this assay labeled with biotin (Biotin Probe) or unlabeled as self-competition (Competition) Lane 1—free unbound biotin labeled probe is shown and is indicated by an open arrow head. Lane 2—contains HeLa nuclear lysate, a shift is indicated by closed arrowhead. Lanes 3–5—self-competition with unlabeled probe at increasing concentrations. c The [−71 to −98] probe (Test), two control probes (Can and SC) and the Modified [−71 to −98] (Mod) probes were used in this assay. Lanes 1–3 positive shifts for the [−71 to −98] probe as well as two positive control probes designed to bind EGR1 (Table 3). Lane 4—[−71 to −98] modified probe, four nucleotides in the EGR1 cluster region were modified and the shift is abolished. d The [−71 to −98] probe was used in this assay (Biotin Probe). Lane 1—shows free unbound biotin labeled probe which is indicated by the open arrow head. Lane 2—contains HeLa nuclear lysate and recapitulates the shift from 6B/C. There are three binding populations in the shift indicated by closed arrow heads (I, II, III). Lane 3—reaction contains polyclonal IgG antibody targeting EGR1 along with HeLa nuclear lysate. Addition of the anti-EGR1 abolishes the binding of the second population to the probe (II). Lane 4—reaction contains HeLa nuclear lysate and polyclonal IgG antibody targeting ATP2B2. This antibody does not abolish the II shift and acts as a negative control for the supershift observed in Lane 3. e Average pixel density of the standard shift was normalized to the average pixel density of the test shift for each experiment (self-competition, modified EGR1 probe assay and supershift assay). White boxes indicate the shifts being compared in each experiment. Normalized values were compared to a theoretical value of 1 using a one sample t test (*P ≤ 0.05 and **P ≤ 0.01). All results are significant