Skip to main content
. Author manuscript; available in PMC: 2012 Jul 12.
Published in final edited form as: Oncogene. 2011 Jun 20;31(2):149–160. doi: 10.1038/onc.2011.222

Fig. 3. Interaction between Nanog, Stat-3 and the upstream promoter/enhancer region of miR-21 promoter in HSC-3 cells.

Fig. 3

A: In vivo binding of Nanog and Stat-3 (or phosphorylated Stat-3) to the miR-21 upstream promoter/enhancer region in HSC-3 cells: ChIP assay was performed in HSC-3 cells following protocols described in the Materials and Methods using the Stat-3 binding site-containing miR-21 promoter (upstream promoter/enhancer region)-specific primers by PCR. Identical volumes from the final precipitated materials were used for the PCR reactions [untreated cells (lane 1); cells treated with HA for 30min (lane 2); cells pretreated with anti-CD44 antibody for 1h plus 30min HA addition (lane 3); cells pretreated with scrambled siRNA with no HA (lane 4); cells pretreated with scrambled siRNA plus 30min HA addition (lane 5); cells pretreated with Nanog siRNA plus 30min HA addition (lane 6); cells pretreated with Stat-3 siRNA plus 30min HA addition) (lane 7). (a: anti-Stat-3-mediated immunoprecipitated material; b: anti-phospho-Stat-3 (pY705)-mediated immunoprecipitated material; c: anti-Nanog-mediated immunoprecipitated material; d: IgG isotype control-mediated precipitated material; e: total input materials). B: Verification of the specificity of Nanog siRNA or Stat-3 siRNA used in the study.

Cell lysates isolated from HSC-3 cells [transfected with Nanog siRNA or Stat-3 siRNA-target or scrambled siRNA] were solubilized by 1% Nonidet P-40 (NP-40) buffer followed by immunoblotting with anti-Stat-3 antibody (a), anti-Nanog antibody (b) or anti-actin (a loading control) (c).