Normal mouse immunoglobulin g igg

Chromatin immunoprecipitation (ChIP) assays were performed according to manufacturer’s instructions (Magna ChIP G Chromatin Immunoprecipitation; Millipore). Briefly, 3 × 10⁶ SK-hep-1 cells were stimulated for 3 h with 20 μM of LiCl, cross-linked with 1% formaldehyde for 10 min at room temperature, lysed and sonicated to average lengths of 200- to 1000-bps. After centrifugation, the samples were diluted 10x to assess input DNA. Anti-TCF-4 (Millipore), normal mouse immunoglobulin G (IgG) (Millipore), and 5 μg of monoclonal anti-β-catenin antibody (BD Transduction Laboratories) were added and incubated with gentle agitation overnight at 4 °C. The DNA-protein complexes were eluted and incubated with proteinase K at 62 °C for 2 h to reverse the formaldehyde cross-link. The primers used in the ChIP assays are listed in Supplementary Table 2B.

Anti-NRF2 (sc-13032), anti-Lamin B (sc-6217), anti-Pol II (sc-899), anti-HSP90 (sc-59577) and anti-BACH1 (sc-14700) antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-β-Actin antibody (A1978) was obtained from Sigma-Aldrich. Anti-Pol II antibody (clone CTD4H8) and normal mouse immunoglobulin G (IgG) were purchased from Millipore. Anti-HO-1 (ab68477) was purchased from Abcam.

The lysis of SUNE-1 cells was done using the EZMagna RIP kit (Millipore, USA) as per the manufacturer’s protocol and then incubated with RIPA buffer containing magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody (Millipore). Normal mouse Immunoglobulin G (IgG) (Millipore) was used as an NC. There was the incubation of the sample with protease K, the immunoprecipitated RNA extraction, and RT-qPCR analysis of the purified RNA.