Erα hc 20 antibody sc 543

For nuclear extract, 5 × 10⁷ control MCF7 cells or cells transfected with HA-Api5 (with Hemagglutinin tag) expression vector were washed in PBS and resuspended in 4 ml of fractionation buffer (0,15M NaCl;10 mM MgCl2; 10 mM CaCl2; 15 mM Tris, pH 7.5; 0.1% Tween 20; proteases inhibitors). Cells were disrupted by freezing/thawing. Nuclei were collected by centrifugation resuspended in Lysis Buffer (150 mM NaCl, 1% 100X Triton,50 mM Tris HCl pH = 8) and sonicated. Co-Immunoprecipitation were performed using the mMACS HA Tagged Protein Isolation Kit (Miltenyi Biotec) or the BioAdembeads protein G 0433 kit for endogenous co-immunoprecipitation. Western Blot were performed as described with API5 antibody (ab56392 Abcam), and ERα HC-20 antibody: sc-543 (Santa Cruz), and monoclonal anti-HA antibody H9658 (Sigma) and anti-RPS19 (3C6 Abnova).

Cells were collected, resuspended in sample buffer and sonicated according to Sambrook et al. [34 ]. 30 μg of proteins were resolved in 4–20% denaturing polyacrylamide gels (Thermo Scientific) and transferred onto a nitrocellulose membrane (Amersham). Immunoblotting were performed using polyclonal anti-API5 antibody (ab56392 Abcam), ERα HC-20 antibody: sc-543 (Santa Cruz), monoclonal anti-HA antibody H9658 (Sigma) anti-RPS19 (3C6 Abnova), anti-ERK1/2 (#4696 Cell signaling) anti-phospho-ERK1/2 (#9106 Cell signaling). Secondary antibodies anti mouse HRP (#7074) and anti rabbit HRP (#7076) were from Cell Signaling. The signal was detected using enhanced chemoluminescence detection reagent Clarity (BioRad Laboratories). Signal was registered with a CCD camera (Vilber Lourmat).