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. 2013 Jun 26;305(5):F653–F662. doi: 10.1152/ajprenal.00177.2013

Fig. 7.

Fig. 7.

Reciprocal effects of Sp1 and Dot1a/Af9 on αENaC promoter occupancy and activity are independent. A: chromatin from mIMCD3 cells stably transfected with pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or the Sp1 binding site mutant pcDNA3.1-Zeo-1.3ENaCαΔSp1+222/+229-Luc (ΔSp1+222/+229) was immunoprecipitated with the Af9 antibody or IgG. Precipitated R3 subregion segments were quantified by qPCR (n = 3). The value for Af9-IP amplicon/input DNA obtained from pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) was set to 1, and the value from pcDNA3.1-Zeo-1.3ENaCαΔSp1+222/+229-Luc (ΔSp1+222/+229) was normalized to it. The values shown are the mean of triplicate determinations of 3 independent experiments. Error bars indicate ± SE. No statistically significant differences between group means were detected. B: chromatin from mIMCD3 cells stably transfected with pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or the Sp1 binding site mutant pcDNA3.1-Zeo-1.3ENaCαΔSp1+222/+229-Luc (ΔSp1+222/+229) was immunoprecipitated with the Dot1a antibody or IgG. Precipitated R3 subregion segments were quantified by qPCR (n = 3). The value for Dot1a-IP amplicon/input DNA obtained from pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) was set to 1, and the value from pcDNA3.1-Zeo-1.3ENaCαΔSp1+222/+229-Luc (ΔSp1+222/+229) was normalized to it. The values shown are the mean of triplicate determinations of 3 independent experiments. Error bars indicate ± SE. No statistically significant differences between group means were detected. C: mIMCD3 cells stably transfected with pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or the Sp1 binding site mutant pcDNA3.1-Zeo-1.3ENaCαΔSp1+222/+229-Luc (ΔSp1+222/+229) were transiently transfected with a EGFP-mDot1a expression vector or EGFP vector. After 24 h, the firefly activities were measured and normalized to cell protein content. The value obtained for EGFP-transfected cells was set to 1, and that of the pEGFP-mDOt1a-transfected cells was normalized to it. The values shown are the mean of triplicate determinations of 3 independent experiments. Error bars indicate ± SE. No statistically significant differences between group means were detected. D: mIMCD3 cells stably transfected with pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or the Af9 binding site mutant pcDNA3.1-Zeo-1.3ENaCαΔAf9+78/+92 were transiently transfected with empty vector or the Sp1 expression vector pBS-Sp1. After 24 h, the firefly activities were measured and normalized to cell protein content. The value obtained for vector-transfected cells was set to 1, and that of the pBS-Sp1-transfected cells was normalized to it. The values shown are the mean of triplicate determinations of 3 independent experiments. Error bars indicate ± SE. No statistically significant differences between group means were detected.