Skip to main content
. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: Clin Cancer Res. 2014 Feb 11;20(5):1259–1273. doi: 10.1158/1078-0432.CCR-13-1664

Figure 2. Nx inhibits NFκB/Stat3 signaling.

Figure 2

A. Whole cell extracts prepared from Capan-2 or BxPC-3 cells treated with a low, medium, or high dose of Nx for 24 h were subjected to immunoblot analysis with the indicated antibodies.

B. EMSA was conducted with nuclear extracts prepared from Capan-2 or BxPC-3 cells using Stat3 oligonucleotide as the radiolabeled probe in the presence of 100-fold excess of cold competitor (Stat3 oligonucleotide) and heterologous competitor (AP1 oligo), and preincubation with anti-Stat3, anti-p65, both antibodies, or IgG as a negative control. Specific DNA-protein complex formed was labeled with an *. Unbound probe is labeled UB.

C. Whole cell extracts prepared from Capan-2 or BxPC-3 cells treated with low, medium, and high dose of Nx for 24 h were subjected to immunoblot analysis with the indicated antibodies.

D. EMSA was conducted with nuclear extracts prepared from Capan-2 or BxPC-3 cells using NFκB oligonucleotide as the radiolabeled probe in the presence of 100-fold excess of cold competitor (NFκB oligonucleotide) and heterologous competitor (AP1 oligo), preincubation with anti-Stat3, ant-p65 or both antibodies, and IgG as a negative control. Specific DNA-protein complex formed was labeled with an *. Unbound probe is labeled UB.

E and F. Cellular localization of Stat3 (green) and p65 (red) was observed by confocal microscopy after immunofluorescent staining of stable scrambled and Stat3 knockdown transfectants of Capan-2 (E) and BxPC-3 (F) cells that were treated with Nx. Capan-2 and BxPC-3 cells treated with Nx were stained with immunofluorescent antibodies for Stat3 (green) and p65 (red) and cellular localization of proteins observed by confocal microscopy.