Protein isolation and immunoblotting

Protein levels were assessed by Western blot in mammary glands and tumors. Total protein was extracted from tissues using RIPA lysis buffer (0.1% SDS, 0.5% Sodium Doxycholate, 1% NP-40, 1 mM EDTA, 1 mM sodium orthvandate, 1 mM PMSF, 5 mM pyrophosphate, 10 mM glycocerophosphate, 50 mM Tris-HCl pH 7.4, 150 mM NaCl) supplemented with Mini Complete Protease Inhibitor (Roche, Germany). Protein concentration was measured using the BCA Protein Assay kit (Thermo Scientific) per manufacturer’s protocol. Protein extracts were separated on a 4–12% gradient denaturing poly-acrylamide gel (SDS-PAGE). Proteins were then transferred to a nitrocellulose membrane using the Invitrogen iBlot 7-min Blotting System. This was followed by blocking with Tris buffered saline + Tween 20 (TBST) plus 5% nonfat dry milk and incubation with specific primary antibodies (1:1000) overnight at 4 °C (PERK: 3192-Cell Signaling; HIF-1α: NB 100–134-Novus Biologicals; p62: 5114-Cell Signaling; NFκB p65: sc-8008- Santa Cruz Biotechnology, DNMT1: 5032-Cell Signaling; DNMT3a: 2160-Cell signaling; HDAC1: 10197–1-AP-Proteintech; ERα: 21244-1-AP-Proteintech; ERβ: 14007-1-AP-Proteintech). After several washes in TBST, membranes were incubated with secondary antibody at room temperature for 1 hour. Membranes were developed using HyGLO Chemiluminescent HRP antibody detection spray and developed on an Amersham imaging system or exposed to Kodak autoradiography films. Protein levels were determined by band intensity using Quantity One software (Bio-Rad) and the target proteins were normalized by β-actin (1:1000, sc1616-Santa Cruz Biotechnology) or Cyclophilin (1:1000, 2175-Cell Signaling)