Sc 544

For Western blot assay, cells were harvested, washed with PBS, boiled for 10 minutes in lysis buffer (125 mmol/L Tris HCl pH 6.8, 5% of SDS) and supplemented with protease inhibitors (11873580001, cOmplete, Roche). Samples were then sonicated for 20 seconds, followed by centrifugation for 15 minutes at 13,000 rpm and quantified with micro BCA Assay kit reagent (23235, Thermo Fisher Scientific) using the Spark microplate reader (TECAN). Proteins were separated by SDS-PAGE on precast 4% to 12% Bis-Tris NuPAGE gels (NP0321PK2, Thermo Fisher Scientific) and blotted to Immobilon PVDF membranes (IPVH00010, Merck Millipore) using the XCell II blot module (EI9051, Thermo Fisher Scientific). Membranes were then saturated for 1 hour in Tris-buffered saline containing 5% BSA and incubated overnight with the antihuman primary antibodies recognizing: JMJD6 (ab64575, Abcam and P1495, Sigma-Aldrich), Actin-β (A1978, Sigma-Aldrich), ERα (sc-544, G20, Santa Cruz Biotechnology and MA1–80216, Invitrogen), ANXA1 (ab214486, Abcam) and Vinculin (V9131, Sigma-Aldrich). After hybridization with the appropriate secondary antibody (anti-rabbit and anti-mouse IgG, GE HealthCare), proteins were detected by chemiluminescence (EuroClone).