Horseradish peroxidase hrp conjugated goat anti mouse secondary antibody

HL-1 or NIH3T3 cells co-transfected with pcTnT-tTSTA-fluc and pCMV-hRL and exposed to raloxifene were harvested by trypsinization, lysed by sonication in RIPA buffer (Cell Signaling, Danvers, MA), and centrifuged to obtain the supernatant, whose protein content was determined using the Bio-Rad protein assay. Ten micrograms of protein from each sample was resolved by 4–12 % gradient SDS/PAGE (Invitrogen) and electroblotted to a 0.2-μm nitrocellulose membrane (Schleicher & Schuell, Keene, New Hampshire). The membrane was subsequently blocked with 5 % nonfat dry milk in TBS containing 0.01 % Tween 20 (TBST buffer) for 3 h and probed overnight at 4 °C on a rotating platform with mouse monoclonal anti-human ERα antibody (Santa Cruz, Santa Cruz, CA). Afterwards, the blot was incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Cell Signaling) for 2 h at room temperature and further developed using the LumiGlo enhanced chemiluminescence substrate (Cell Signaling) following the manufacturer’s protocol. Afterwards, the blot was stripped and reprobed first with goat polyclonal anti-human cardiac troponin T antibody (Santa Cruz) and later with mouse anti-human β-actin antibody (Sigma-Aldrich) using HRP-conjugated donkey anti-goat secondary antibody (Promega) and HRP-conjugated goat anti-mouse secondary antibody (Cell Signaling), respectively.