Ab32574

Total protein lysates were obtained by lysing the cells with ice –cold RIPA buffer supplemented with phosphatase inhibitor cocktail (Roche, Canada). The quantification of protein was measured using the Bradford assay (Sigma, USA). Next, 100 μg of each sample proteins were subjected to a 10 % SDS-Page before the proteins were transferred onto a PVDF membrane (Roche, Canada) using the Pierce Fast Semi-Dry Blotter (Pierce, USA). Afterwards, the membranes were blocked in 0.5 % skimmed milk overnight. The following day, the membranes were washed in TBST for 10 min to a total of 3 washes and were incubated with the antibodies, anti-SNAIL (ab82846, Abcam, USA), anti-CXCR4 (ab2074, Abcam, USA), anti-VEGF (ab1316, Abcam, USA), anti-COX2 (ab15191, Abcam, USA), anti-NFKB (AHP 1342, Abdserotec, USA), anti-MRP1(ab32574, Abcam, USA). Afterwards, the membranes were incubated in the appropriate secondary antibodies conjugated with HRP. The western blots were then developed under chemiluminescence condition (SuperSignal West Pico, Pierce, USA) using the ChemiDocXRS machine (Bio-rad, USA). The bands were then analyzed using the Quantity One 1D Analysis software (Bio-rad, USA).

Total proteins were extracted using radio‐immunoprecipitation assay (RIPA) lysis buffer (P0013B; Beyotime Biotechnology Co., Ltd.) containing phenylmethylsulfonyl and phosphatase inhibitors. After a 30‐min incubation on ice, the samples were centrifuged at 12 000 rpm for 10 minutes at 4°C, followed by the supernatant collection. A bicinchoninic acid (BCA) kit (Beyotime Biotechnology Co., Ltd.) was utilized to determine protein concentration. Next, 30 µg protein was separated by sodium dodecyl sulfonate‐polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane using wet transfer apparatus. After that, the membrane was blocked with 5% skimmed milk in Tris‐buffered saline Tween (TBST) buffer for 1.5 hours and incubated with the primary antibody to MRP1 (1:500; ab32574, Abcam) at 4°C overnight. On the following day, the membrane was further incubated with the secondary antibody, horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit antibody to immunoglobulin G (IgG; 1:2000‐50000; ab205718) for 2 hours at room temperature. Subsequently, the protein bands were visualized with an enhanced chemiluminescence (ECL) reagent, which were imaged on SmartView Pro 2000 (UVCI‐2100; Major Science). Image analysis was followed using Quantity One software. The target proteins were quantified as relative gray values against the internal reference GAPDH.

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).