Anti p akt

Cells were lysed with RIPA buffer (BIOSESANG, Sungnam, Korea) containing a protease inhibitor cocktail (AMRESCO, Solon, OH, USA) and incubated on ice for 20 min. Proteins were obtained via centrifugation at 15,000 × g for 30 min and separated using SDS-PAGE and transferred to membranes. The membranes were blocked with 5% skim milk at room temperature (RT) for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-AURKA (Invitrogen), anti-p-AKT, anti-p-ERK, anti-XCL1 (R&D, Minneapolis, MN, USA), antiprotein kinase B (AKT), antiextracellular signal-regulated kinase (ERK), antipoly ADP ribose polymerase (PARP), anticleaved caspase-3, antifocal adhesion kinase (FAK), anti-p-FAK, anti-c-Jun N-terminal kinases (JNK), anti-p-JNK (Cell Signaling Technology, Danvers, MA, USA), antiannexin V, antifibronectin (Abcam, Cambridge, MA, USA), anti-DOCK2, and anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies at RT for 1 h. The membranes were then washed with TBST, and protein bands detected using a gel imaging system (Amersham Imager 600, GE Healthcare, Buckinghamshire, UK). Band intensities were measured using ImageJ (National Institutes of Health (NIH), Bethesda, MD, USA) and then normalized to that of β-actin.

S-allyl cysteine was purchased from Abcam. Trypan blue, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA),5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), and Wortmannin were purchased from Sigma-Aldrich, USA. Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA solutions were purchased from HiMedia, India. INTERFERin siRNA transfection reagent was purchased from Polyplus, USA. Taq polymerase and dNTPs were purchased from Thermo Fisher Scientific, USA. Random hexamer primer and RiboLock RNase inhibitor, and RevertAid reverse transcriptase were purchased from Thermo Scientific, USA. Anti-α-tubulin antibody was purchased from BioBharati LifeScience Pvt. Ltd., India. Anti-Nrf-2, anti-Akt, anti-p-Akt, Bcl2, Bax, caspase 3, and cytochrome c antibodies were purchased from R&D systems, USA. Primers for real time PCR were purchased from Integrated DNA Technologies, USA.

Protein extracts were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Germany). Protein expression was detected by western blot analysis. Antibodies used herein including anti-IL-6, anti-TNF-α, anti-p-AKT, anti-AKT, anti-SRC, anti-p-SRC, anti-VEGFA, anti-MAPK1, anti-IL-1β, anti-EGFR and β-actin were obtained from R&D Systems. Band density was quantified using ImageJ software and normalised to the corresponding control group.