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. Author manuscript; available in PMC: 2013 Dec 1.

Published in final edited form as: J Mol Endocrinol. 2013 Apr 23;50(3):361–374. doi: 10.1530/JME-12-0154

siRNA silencing of Egr-1 reverses 13cRA mediated down-regulation of AT1_AR. (A) Western blot analysis of cells treated with or without 13cRA and transfected with negative control scrambled siRNA oligonucleotides or Egr-1 targeted siRNA oligonucleotides. (B) Immunofluorescent microscopy using anti-Egr-1 to confirm effective silencing of 13cRA induced Egr-1. Egr-1 antibody labeled with FITC (green) and nuclei are stained with DAPI (blue). (C) AT1_AR mRNA expression restored after Egr-1 knockdown (-out) (D). Radio-labeled ligand binding assay showing restoration of binding of AngII to membrane receptors after Egr-1 knockout. All data are expressed as mean ± SEM. “(−)Ctrl” and “(−)RA” indicate cells that are transfected with scrambled negative control siRNA. Egr-1 imaging indicates 13cRA treatment for 60 min while AngII binding and mRNA studies reflect 20 hour 13cRA treatment. (E) Accompanying mobility shift in selective gene knockout studies included. Samples were analyzed on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. The position of the protein-DNA complex is indicated by arrow. Labeled probe in the absence of nuclear extract (lane 1), in the presence of untreated nuclear extract (lane 2), in the presence of 13cRA exposed nuclear extract (lane 3), nuclear extracts of both untreated and treated with 13cRA after siRNA mediated silencing of Sp1 (lanes 4 and 5, respectively), and nuclear extracts of both untreated and treated with 13cRA after siRNA mediated silencing of Sp1 (lanes 6 and 7, respectively).

siRNA silencing of Egr-1 reverses 13cRA mediated down-regulation of AT1_AR. (A) Western blot analysis of cells treated with or without 13cRA and transfected with negative control scrambled siRNA oligonucleotides or Egr-1 targeted siRNA oligonucleotides. (B) Immunofluorescent microscopy using anti-Egr-1 to confirm effective silencing of 13cRA induced Egr-1. Egr-1 antibody labeled with FITC (green) and nuclei are stained with DAPI (blue). (C) AT1_AR mRNA expression restored after Egr-1 knockdown (-out) (D). Radio-labeled ligand binding assay showing restoration of binding of AngII to membrane receptors after Egr-1 knockout. All data are expressed as mean ± SEM. “(−)Ctrl” and “(−)RA” indicate cells that are transfected with scrambled negative control siRNA. Egr-1 imaging indicates 13cRA treatment for 60 min while AngII binding and mRNA studies reflect 20 hour 13cRA treatment. (E) Accompanying mobility shift in selective gene knockout studies included. Samples were analyzed on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. The position of the protein-DNA complex is indicated by arrow. Labeled probe in the absence of nuclear extract (lane 1), in the presence of untreated nuclear extract (lane 2), in the presence of 13cRA exposed nuclear extract (lane 3), nuclear extracts of both untreated and treated with 13cRA after siRNA mediated silencing of Sp1 (lanes 4 and 5, respectively), and nuclear extracts of both untreated and treated with 13cRA after siRNA mediated silencing of Sp1 (lanes 6 and 7, respectively).