Byl40422

The expression of apoptosis-related proteins was evaluated by Western blot analysis. Using RIPA buffer containing protease and phosphatase inhibitors (Solarbio, R0010, Beijing, China), the total proteins were extracted from treated ZR-75-30 and BT474 BC cells. After quantification with the BCA Kit (Thermo, PICPI23223), 25 μg of proteins were separated using 10% SDS-polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, HATF00010, USA). Subsequently, the membranes were blocked with 5% skim milk (BD Biosciences, BYL40422) for 1 h, followed by incubation in primary antibodies against IQGAP3 (1: 1000, Abcam, Ab88353), ERK1/2 (1: 1000, Cell Signaling Technology [CST], #9102), p-ERK1/2 (1: 1000, CST, #9101), Bax (1: 1000, Abcam, Ab32503), Bcl2 (1: 1000, Abcam, Ab196495), and GAPDH (1: 2000, CST, #5174) overnight at 4°C. The next day, at room temperature, the second antibodies labeled with HRP (1: 1000, Beyotime, Shanghai) goat anti-rabbit (A0208) and goat anti-mouse (A0216) antibodies were used to incubate the membranes for 2 h. The blots were developed by 5-min incubation with chemiluminescent reagent (Millipore, WBKLS0100), and then visualized on an ECL imaging system (Tanon, Tanon-5200, Shanghai). The protein expression, normalized to GAPDH, was analyzed and calculated by version 1.47v Image J software (Bethesda, MD, USA).

Total protein was extracted, and the protein concentration was quantified using a BCA protein assay kit. The proteins were separated by 10% SDS‐PAGE, followed by semidry transfer to polyvinylidene fluoride (PVDF) membranes (HATF00010; Millipore). After blocking in 5% skim milk (BYL40422; BD Biosciences) for 1 hour at room temperature, the membrane was incubated with anti‐DDAH1 (1:1000; Ab180599; Abcam), anti‐NRF‐2 (1:1000; Ab62352; Abcam), anti‐VE‐cadherin (1:1000; Ab166715; Abcam), anti‐PRMT (1:1000; Ab190892; Abcam), anti‐Vimentin (1:1000; ab8925; Abcam), anti‐ERK1/2 (1:1000; #4695; Cell Signaling Technology [CST]), anti‐p‐ERK1/2 (1:2000; #4370; CST), anti‐MMP9 (1:1000; Ab76003; Abcam) and anti‐GAPDH (1:2000; #5174; CST) at 4°C overnight. Then, a horseradish peroxidase‐conjugated secondary antibody (1:1000; Beyotime) was added. After developed by using a chemiluminescent reagent (WBKLS0100; Millipore) for 5 minutes, the protein bands were visualized on an ECL imaging system (Tanon‐5200; Tanon). Western blot analysis was performed as previously described.⁵¹

After 48 h of infection, DU145 or PC-3 cells were harvested to lyse in RIPA buffer (Solarbio, R0010, Beijing, China) complemented with protease and phosphatase inhibitors, and incubated on ice for 30 min to fully homogenize. After centrifuging at 12 000 g at 4°C for 10 min, the protein in the supernatant was collected and quantified by a BCA protein kit (Thermo Fisher Scientific, PICPI23223). Portions of protein were separated by 10% SDS-PAGE electrophoresis, and then transferred semi-dry to polyvinylidene fluoride (PVDF) microporous membrane (Millipore, HATF00010, Bradford, MA, USA). The blots were blocked in 5% skim milk (BD Biosciences, BYL40422, USA), followed by incubation with primary antibodies against NFATc1 (1: 1000, Cell Signaling Technology [CST], #8032, Danvers, MA, USA), c-myc (1: 1000, Abcam, Ab39688, Cambridge, MA, USA), PKM2 (1: 1000, Abcam, Ab38237), GAPDH (1: 2000, CST, #5174) at 4°C with gentle shaking overnight. The next day, after washing 5–6 times, the blots were incubated with corresponding secondary antibodies (1: 1000, Beyotime, Shanghai, China) at room temperature for 1 h. After washing again and incubation with chemiluminescent reagent (Millipore, WBKLS0100) for 5 min, the blots were visualized by an ECL imaging system (Tanon, Tanon-5200) and the protein levels were calculated by Image J software (version 1.47v, Bethesda, MD, USA).