Anti akt

Cell samples were lysed in NP40‐containing buffer with PMSF, protease inhibitors, and phosphatase inhibitors. Individual cell lysate samples (30 µg/lane) were separated via SDS‐PAGE on a 12% resolving gel and electro‐transferred to polyvinylidene difluoride membranes (Millipore, Hong Kong). The membranes were blocked with 5% non‐fat dry milk in TBST and incubated overnight at 4°C with primary antibodies, including anti‐human LIPH (1:500, Proteintech, Wuhan, China), anti‐GAPDH (Cell Signaling Technology, Danvers, MA), anti‐CD44 (60224‐1‐Ig), anti‐CD24 (10600‐1‐AP), anti‐Oct4 (11263‐1‐AP), anti‐Sox2 (11064‐1‐AP), anti‐vimentin (WL01960), anti‐MMP2 (WL01579a, WanleiBio, China), anti‐FAK p397 (ab24781, Abcam, UK), anti‐Integrin‐ß3 (WL02735b, WanleiBio), anti‐p‐AKT (WLp001, WanleiBio), anti‐AKT (WL0003b, WanleiBio), anti‐RAS (WL0257, WanleiBio), anti‐CAPN2 (abs137284), anti‐Paxillin (WL00415, WanleiBio), anti‐Vinculin (abs131199), and anti‐Tubulin‐α (2144s, CST, USA). The bound antibodies were detected with horseradish peroxidase (HRP)‐conjugated secondary antibodies (1:10 000). The immunoblotting signals were visualized using enhanced chemiluminescent reagents. The ratios of target proteins to control GAPDH were determined by densitometric analysis using the ImageJ software application (NIH, Bethesda, MD, USA).

Briefly, approximately 20 μg of protein was loaded into each gel well resolved by 10% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyvinylidene fluoride (PVDF) membranes were incubated overnight at 4 °C in tris buffered saline (TBS) containing 3% nonfat dry milk. Anti-Lyn, Anti-TNF-α, Anti-Akt, Anti-p-Akt and Anti-NF-κB p65 antibody were procured from WanleiBio, China. The blot was then washed and incubated with secondary antibody (WanleiBio, China). Antibody binding was detected by chemoluminescence staining using an electrochemiluminescence (ECL) detection kit (Beyotime Biotechnology, China). The density of each band was quantified by densitometry with Gel-Pro-Analyzer softweare.

Protein extracts were obtained from cell lysates and quantified using the bicinchoninic acid (BCA) analysis method (Wanleibio, Shenyang, China). Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. (Merck Millipore, Billerica, MA, USA). After incubation with high-affinity antibodies (anti-p21 (1 : 500), anti-PTEN (1 : 500), anti- AR (1 : 500), anti-mTOR (1 : 500), anti-p-mTOR (1 : 500), anti-AKT (1 : 500), anti-p-AKT (1 : 500), and anti-β-actin antibody (1 : 1000) (Wanleibio, Shenyang, China), the membranes were incubated with a secondary antibody (1 : 5000, Wanleibio, Shenyang, China). After incubation, ECL luminescent solution (Wanleibio, Shenyang, China) was evenly added to the PVDF membranes, these membranes were loaded onto the cassette and exposed in a darkroom. After washing, signals were detected and analyzed using the Gel-Pro-Analyzer software.