Total mek

Cell pellets were lysed in RIPA buffer (50 mM Tris pH7.4, 140 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate) containing phosphatase and protease inhibitor tablets (Roche). Cell pellets were lysed for 20 minutes on ice, after which nuclear and cellular debris was removed by centrifugation at 13,000 RPM for 10 minutes at 4°C. 10–20 μg of protein lysates were electrophoresed on NuPage precast gels (Invitrogen) and immunoblotted with anti-NF1 (Bethyl Antibodies), anti-phospho-SRC (Tyr416) (Cell Signalling Technology [CST]), anti-SRC (CST), phospho-ERK (CST), phospho-MEK (CST) total ERK (Cell signaling Technology), total MEK (CST), phospho-HER2 (CST), total HER2 (Santa Cruz), phospho-Insulin Receptor/IGF1R (CST), total insulin receptor (CST), phospho-FAK (Tyr925) (CST), total FAK (CST). As loading controls, vinculin (Santa Cruz) and beta-actin (Sigma) were used. Membranes were then incubated in anti-IgG-HRP (CST) and chemiluminescent detection (Luminata Cresendo, Millipore).

All of the chemicals/reagents were purchased from different authorized companies. Most of the cell culture-based chemicals and reagents were purchased from Sigma-Aldrich, and the cultureware was purchased from the Nunclon Thermo-Scientific-India. The Western blot apparatus, tools, and reagents were purchased from Bio-Rad Technologies, USA. Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin, propidium iodide (PI), 2,7-H₂DCF-DA, proteinase K, ribonuclease-A, agarose, dimethyl sulfoxide (DMSO), Dulbecco’s phosphate buffer saline (DPBS), fetal bovine serum (FBS), LC3I/II, and anti-β-actin antibodies were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Antibodies against caspase-3/caspase-8, B-cell lymphoma 2 (Bcl-2), Bcl-2 homology 3-only protein (Bim), BCL2-associated X (Bax), ATF4, phospho-mitogen-activated protein kinase kinase (MEK), phospho-extracellular signal-regulated kinase (ERK), total ERK, total MEK, phospho-p38, p-38, phospho-JNK, JNK, phospho-AKT, nuclear factor erythroid 2-related factor 2 (Nrf-2), catalase, copper/zinc superoxide dismutase (Cu/Zn SOD), AKT, Beclin1, P62, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology, Inc. Glycyrrhizin was purchased from Sigma-Aldrich.