Anti p65 antibody d14e12

DU145 cells were treated with 2D5 peptide at the indicated concentrations and for the indicated periods and then lysed in lysis buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% deoxycholate, and 0.1% SDS) with sonication. After centrifugation, the supernatants were subjected to SDS-PAGE and then immunoblotted. The following antibodies were used for immunoblotting: anti-Myc antibody (9E10, Sigma), anti-HA antibody (C29F4, Cell Signaling Technology), anti-EGFR antibody (D38B1, Cell Signaling Technology), anti-phospho-EGFR Y1068 antibody (Cell Signaling Technology), anti-GST antibody (Z-5, Santa Cruz Biotechnology), anti-STAT3 antibody (79D7, Cell Signaling Technology), anti-phospho-STAT3 antibody (GeneTex), anti-ERK antibody (K-23, Santa Cruz Biotechnology), anti-phospho-ERK antibody (Cell Signaling Technology), anti-β-actin antibody (AC-15, Santa Cruz Biotechnology), anti-p65 antibody (D14E12, Cell Signaling Technology), anti-phospho-p65 antibody (93H1, Cell Signaling Technology), and anti-phosphotyrosine antibody (4G10, GeneTex).

The following antibodies were used at the specified dilutions: anti‐p65 antibody (D14E12; Cell Signaling Technology, Danvers, MA, USA; 1 : 400) and Alexa Fluor‐conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA USA; 1 : 1000).

Chromatin immunoprecipitation (ChIP) assay was performed with pierce agarose ChIP kit (Pierce) according to the manufacturer’s instructions. In brief, 1 × 10⁷ of bone marrow cells were fixed and immunoprecipitated with anti-p65 antibody (D14E12; Cell Signaling) or rabbit IgG (Pierce). Immunoprecipitated DNA fragment were quantified by real-time PCR with the use of the following primers, which amplify the Junb enhancer region containing NF-κB binding sites; forward 5′-ATAAGGTTCAGTACAAACGCCC-3′, reverse 5′-GCGTCACTGAGCTGAATAGG-3′. Fold enrichment was normalized to rabbit IgG-precipitated samples.