Anti β actin

MDA-MB-231 and MCF-7 cells were lysed in RIPA lysis buffer on ice for 30 min, then centrifuged (12000 g/min; 30 min) at 4°C. A bicinchoninic acid (BCA) assay was used to detected protein concentrations. Equal amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (millipore, USA), and then incubated with primary antibodies overnight at 4°C after the membranes were blocked (5% skim milk in PBS with 0.1% Tween 20) for 4 h. The next day, the membranes were imaged with gel imaging equipment (Bio-Rad, USA) after the membranes were incubated with secondary antibodies for 2 h. β-actin was used as a loading control. The following antibodies were used: Bcl-2 and Bax (Cell Signaling technology, USA); anti-RIP1, anti-RIP3, and p-RIP3 (Santa Cruz Biotechnology, USA); TNF-α (Abcam, USA); Caspase 3 (Enzo, USA); Ppm1b (BETHYL, USA); anti-β-actin (Biosharp, China) All reagents were dissolved according to the manufacturer’s instructions.

RA-FLSs were treated with different concentrations of shikonin for 24 h and then collected. Total cell lysates were prepared using RIPA buffer supplemented with protease inhibitors (Roche, Shanghai, China) and PMSF (Sigma, USA). Protein concentrations were determined with BCA kits (Beyotime, Shanghai, China). The cells were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk, the PVDF membranes were incubated with their specific primary antibodies in TBST at 4 °C with primary antibodies recognizing PKM2 (1:1000, Cell Signaling Technology, USA), GULT1, HK2, PI3K, p-PI3K (1:1000, Santa Cruz Biotechnology, USA), AKT, p-AKT (1:1000, Abcam, USA), mTOR, BAX (1:1000, Cell Signaling Technology, USA), Bcl-2 (1:1000, Cell Signaling Technology, USA), caspase 3 (1:1000, Enzo, USA), LC3 (1:1000, Santa Cruz Biotechnology, USA), and anti-β-actin (1:1000, Biosharp, China). All reagents were dissolved according to the manufacturer’s instructions.
After three washes with TPBS, the secondary antibody (1:5000) was added followed by incubation at room temperature for 1 h. Proteins were visualized and detected by enhanced chemiluminescence detection reagents (Pierce, Thermo Fisher Scientific) and analyzed with an Image Quant LAS 4000 imaging system (GE Healthcare, Pittsburgh, PA, USA).