Electrochemiluminescent detection system

Total protein of resistant PCa cell lines was extracted with RIPA lysis buffer (Beyotime, Shanghai, China). Total protein was separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then, the PVDF membranes were blocked in tris buffered saline tween (TBST) buffer with 5% non-fat dry milk for 1 h. Afterward, the membranes were incubated with primary antibodies at 4 °C overnight. After washing with TBST, the PVDF membranes were incubated with secondary antibodies: goat anti-rabbit IgG-HRP (ab6721, 1:10,000, Abcam, MA, USA) or goat anti-mouse IgG-HRP (ab205719, 1:5000, Abcam) for 1 h at 37 °C. The blots were determined using the electrochemiluminescent detection system (Thermo fisher scientific). The primary antibodies used were listed as below: anti-CyclinD1 (ab16663, 1:200, Abcam), anti-matrix metalloproteinase 9 (MMP9, ab76003, 1:1000, Abcam), anti-Vimentin (ab92547, 1:1000, Abcam), anti-E-cadherin (ab15148, 1:500, Abcam), anti-GAPDH (ab8245, 1: 500, Abcam), anti-Cleaved-caspase-3 (Cleaved-casp-3, ab2302, 1:1000, Abcam), anti- anti-Cleaved-caspase-9 (Cleaved-casp-9, ab2324, 1:2000, Abcam), anti-Multidrug Resistance associated Protein 1 (MRP1, ab32574, 1:500, Abcam), and anti-multidrug resistance-1 (MDR1, Ab170904, 1:1000, Abcam).