Nuclear and cytoplasmic isolation kit

Total proteins were extracted with RIPA buffer (Thermo Scientific, USA) and 1 mM PMSF. Cytoplasmic and nuclear subcellular fractionation was performed according to the manufacturer’s instructions using the Nuclear and Cytoplasmic Isolation Kit (KeyGEN, China). All proteins were separated on 10% SDS-PAGE (Invitrogen, USA) gels, transferred onto PVDF membranes (Millipore, USA), and then blocked using 5% skim milk. The proteins were detected by incubating the following primary antibodies: anti-AR (AR441; Abcam, USA), anti-estrogen receptor (ER; D8H8; CST, USA), anti-progesterone receptor (PR; 6A1; CST, USA), anti-HSP27 (G3.1, Abcam, USA), anti-HSP27 (phospho S15) (EP2293Y, Abcam, USA), anti-HSP27 (phospho S78) (Y175, Abcam, USA), anti-HSP27 (phospho S82) (EPR7278, Abcam, USA), anti-β-actin (8H10D10, CST, USA), and anti-Histone H3 (D18C8, CST, USA) overnight; and incubated with horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG, followed by detection using the ECL detection kit (Solarbio, China). Each sample was analyzed in triplicate.

Cellular protein was extracted by RIPA buffer containing PMSF. The Nuclear and Cytoplasmic Isolation Kit (KeyGEN, China) was used for the cytoplasmic and nuclear protein extraction according to the manufacturer’s instructions. The proteins were separated on 10% SDS-PAGE gels, transferred onto PVDF membranes, blocked using 5% skim milk, and incubated with primary antibodies overnight at 4 °C. The protein bands were detected by the ECL detection Kit (Solarbio, China).