Enhanced chemiluminescence kit

The samples were homogenized in 0.1% SDS buffer containing 10 mM EDTA, 125 mM NaCl, 25 mM HEPES, 0.5% deoxycholic acid, 10 mM Na₃VO₄, 0.1% SDS, 1% Triton X-100 with Complete™ protease inhibitor cocktail (Roche, Basel, Switzerland). The cell lysate was centrifuged at 12,000 rpm for 15 min. Then the supernatant-contained protein was collected and the protein concentration was tested by protein assay kit (Bio-Rad, CA, USA). The collected protein was separated on SDS-PAGE gel, and transferred onto PVDF membrane (Millipore, MA, USA). The membrane was blocked with 5% skim milk for 1 h to reduce non-specific binding. Then, the membrane was incubated with one of the following rabbit polyclonal primary antibodies: anti-LC3B-I&II, anti-Beclin-1, anti-p62, anti-β-actin, anti-Tublin, anti-C-myc, anti-N-myc, anti-L-myc, anti-GLS and anti-ERα (Cell Signaling Technology, MA, USA) at 4 °C for 12 h. After 3 times of washes, the blot was incubated with secondary antibody HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology, USA) for 1 h at room temperature. Finally, the signal was detected by the enhanced chemiluminescence kit (Biorbyt, CA, USA) and exposed to X-film