R0010

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).

Proteins were extracted from freshly frozen tissues. Briefly, the tissues were homogenized and lysed with radio immunoprecipitation lysis buffer (Solarbio, R0010, Beijing, China), which has 100 mg/ml of phenylmethanesulfonyl fluoride (ADOOQ, A11901, California, USA) and 1 mg/ml of Aprotinin (ADOOQ, A14263). The lysate was collected and centrifugated for liquid supernatant. A total of 50 μg protein samples were separated with 10% SDS-PAGE (Solarbio, P1040) and transferred onto polyvinylidene fluoride (Millipore, HVLP04700, Arklow, Ireland) membranes. Membranes were blocked at room temperature with 5% skim milk (ACMEC, AC11037, Shanghai, China) powder in TBS (Quartett, 402000192, Berlin, Germany) for 1 h. Membranes were then incubated overnight at 4 °C with primary antibodies against the following proteins: Acyl-CoA synthetase long-chain familymember 4 (ACSL4) (1: 1000, ABclonal, A6826, Wuhan, China), transferrin receptor (TFRC) (1: 1000, ABclonal, A5865, Wuhan, China). Secondary antibodies (1: 1000, ABclonal, AS014, Wuhan, China) were incubated for 1 h at 37 °C and washed three times with PBST (PBS+0.1% Tween 20) (Biomed, PA202, Beijing, China). The protein bands were visualized by Molecular Imager Gel Doc XR System (Bio-Rad, Hertfordshire, UK) and quantified by densitometric analysis using an image analyzer (NIH Image J software, Bethesda, USA).