Goat anti rabbit immunoglobulin hrp

Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2% Triton-X-100, 2 mM EDTA, 0.01% SDS, 50 mM NaF, 0.1 mM Na₃VO₄, 1×Protease Inhibitor/no EDTA), sonicated (20% amp for 15 s) and centrifuged at 15000 rpm for 15 min at 4 °C. 10 µg of cell lysate was denatured and resolved by 6% homemade SDS-PAGE protein gel [49 (link)]. Proteins were transferred onto nitrocellulose membranes and blocked with 10% skim milk 1× Tris-buffered saline, 0.1% Tween 20 (TBST). The membrane was probed with primary antibody; rabbit anti-AR (Santa Cruz, cat #sc-816), mouse anti-ER $\mathrm{\alpha }$ (Santa Cruz, cat #sc-8002), rabbit anti-PGR (Leica Biosystems, # NCL-L-PGR AB), rabbit anti-FOXA1 (Abcam, cat # ab23738), mouse anti-Myc (Sigma Aldrich, cat #M4439), mouse anti-V5 (Thermo Fisher, cat #46-0705), mouse anti-FLAG (Sigma Aldrich, cat #F3165) or rabbit anti-β-Tubulin (Abcam, cat #ab6046) in 1× TBST/2% skim milk overnight at 4°C. The next day, membranes were probed with anti-mouse immunoglobulins/HRP (Dako, cat #P0447) or goat anti-rabbit immunoglobulin/HRP (Dako, cat #P0448) in 1× TBST/2% skim milk, washed with 1× TBST and detected by Clarity Western enhanced chemiluminescence (ECL) substrate (Bio-Rad) using BioRad ChemiDoc MP Imaging System with Imagining Lab 5.0.

The primary antibodies used were as follows: γH2AX (Upstate Technology; 05-636) at 1:800 for IF, RPA (Merck Millipore; LS-C38952) at 1:100 for IF, RAD51 (Santa Cruz Biotechnology; SC-8349) at 1:200 and CENP-F (Abcam; ab108483) for IF, INO80 (Abcam; ab118787, and Bethyl; A303-371A) at 1:2,000 for WB, H2A.Z (Cell Signaling Technology; 2718S) at 1:1,000 for WB, ANP32E (Sigma-Aldrich; SAB2100124) at 1:1,000 for WB, KAP1 (Abcam; ab22553) at 1:1,000 for WB and KU80 (Abcam; ab33242) at 1:1,000 for WB.
The secondary antibodies used were as follows: FITC (Sigma-Aldrich; F0257) at 1:100 for IF, Cy3 (Sigma-Aldrich; C2306) at 1:200 for IF, AlexaFluor 488 (Invitrogen; A21206) at 1:400 for IF, Goat Anti-Rabbit Immunoglobulin/HRP (Dako; P0449) at 1:2,000 for WB, Rabbit Anti-Mouse Immunoglobulins/HRP (Dako; P044801-2) at 1:2,000 for WB.