Red protein g affinity beads

For IP experiments the Chk1 (1:2, E250, Abcam) or GATA3 (1:2, 16E10A23, Biolegend) antibody was incubated with Red Protein G Affinity beads (Sigma-Aldrich) for 1 h at 4 °C on a shaker to allow the antibody to bind to the beads. Cell lysates were collected in RIPA buffer plus inhibitors (protease: complete mini Roche; 11836153001, and phosphatases: cocktail set II Calbiochem; 524625) and then incubated with the antibody-bead mix overnight at 4 °C on a shaker. The following morning, samples were centrifuged, and the supernatant was discarded leaving only the proteins able to bind to Chk1 or GATA3. SDS-polyacrylamide electrophoresis and western blotting of proteins of interest including, GATA3 (1:1000, 16E10A23, Biolegend), PGC1α (1:50, 3G6, Cell Signalling), Nrf1 (2 μg/ml, 492413, R&D Systems), Nrf2 (1 μg/ml, ab89443, Abcam), SOD3 (20 μg/ml, AF3420, R&D Systems), ATR (1:1000, E1S3S, Cell Signalling) and βactin (1:1000, 13E5, Cell Signalling) was then performed. Protein was detected via chemiluminescence by means of the ECL system (GE Healthcare, GERPN2106).

Red protein G affinity beads, RIPA buffer, and protease inhibitor cocktail were purchased from Sigma-Aldrich Corporation (St. Louis, MO). The HSP90 inhibitors, AUY-922 and AT13387, were obtained from Selleck Chemicals (Houston, TX). The BCA Protein Assay Kit was purchased from Pierce Co. (Rockford, IL), and Western blot membranes were purchased from GE Healthcare (Chicago, IL). All antibodies used in Western blots and immunoprecipitation have published immunospecificity data available online. Rabbit anti-AKT, anti-phospho AKT, anti-IKBα, anti-phospho IKBα, anti-STAT3, anti-phospho STAT3, anti-VE-cadherin, anti-occludin, anti-cofilin, and anti-phospho cofilin were purchased from Cell Signaling Technology (Danvers, MA). Mouse monoclonal anti-β-actin and anti-p53 (P8999) were purchased from Sigma-Aldrich Corporation, and secondary IRDye 800CW goat anti-rabbit (926-32211) and IRDye 680RD goat anti-mouse (926-68070) were purchased from LI-COR Biosciences (Lincoln, NE). For SDS-PAGE, Protogel (30% acrylamide mix) and TEMED were purchased from National Diagnostics (Atlanta, GA), Tris-HCl buffer was purchased from Teknova (Hollister, CA), 10% SDS and ammonium persulfate were purchased from Thermo Fisher Scientific, and protein dual-color standards and tricine sample buffer were purchased from Bio-Rad Laboratories.