Ecl blotting analysis system

Cell lysates from A549, 95D, A549-P-S, A549-P, 95D-P-S, 95D-P cells were prepared by extracting proteins with RIPA buffer (Pierce) containing protease inhibitor cocktail set I (Merck KGaA, Germany). The protein concentrations were measured by the BCA protein assay reagent (Pierce Biotechnology, Rockford, IL, USA). An equal amount of total proteins was loaded on each lane and separated by SDS-PAGE and subsequently blotted on a PVDF membrane (pore size, 0.45 am; Millipore, Billerica, MA). Membranes were incubated with blocking buffer for 60 min at room temperature and then incubated with a specific primary antibody against SLC34A2 (Sent Cruz Biotechnology, Santa Cruz, CA, USA), Cycling D3, PI3 Kinas, phospho-Akt (Ser473), Akt, phospho-mTOR (Ser2448), mTOR, phospho-MEK1/2 (Ser217/221), MERK1/2, phospho-p44/p42 MAPK (Erk1/2) (Thr202/Tyr204), Erk1/2 (Cell Signal pathway Technology, Beverly, MA, USA) and GAPDH (Sigma, CA, USA) with Blotto overnight at 4 °C. The membranes were washed and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody. Blots were then exposed to secondary antibodies and visualized by an enhanced chemiluminescence (ECL) blotting analysis system (GE Healthcare Life Sciences, Piscataway, NJ).

Protein samples were isolated from harvested cells using 500 μL radio‐immunoprecipitation assay (RIPA) buffer (Thermo Scientific) with 1 mmol/L phenylmethane sulfonyl fluoride. Then, protein samples were obtained through centrifugation. Protein samples of same volume were subjected to 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (PVDF) (0.45 µm pore size; EMD Millipore). Later, the membranes were blocked by 5% skimmed milk and incubated with primary antibodies at 4°C overnight. GAPDH acted as loading control. Then, the membranes were incubated employing secondary antibody at room temperature for additional 2 hours. Blotting results were analysed applying an ECL blotting analysis system (GE Healthcare Life Sciences). Each trial was repeated at least three times.

The expression of protein was analyzed using Western blot analysis. Whole protein from cells was extracted using M-PER Mammalian Protein Extraction Reagent, containing Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA, USA) and Complete Protease Inhibitor Cocktails (Roche, Lewes, UK) according to the manufacturer’s protocol. Proteins (50 μg) were separated on 7.5% gradient sodium dodecyl sulfate (SDS)–polyacrylamide gels and then transferred electrophoretically to Immobilon-P membranes (Millipore, Billerica, MA, USA). Primary antibodies, including ANXA1 (Cell Signaling Technology, Danvers, MA, USA), phospho EGFR (Tyr1068) (Cell Signaling Technology), EGFR (Santa Cruz, Dallas, TX, USA), Akt (Santa Cruz), phospho AKT (Ser473) (Cell Signaling Technology), phospho HER2 (Tyr877) (Cell Signaling Technology), HER2 (Cell Signaling Technology), cleaved-PARP (Cell Signaling Technology) and β-actin (Sigma, St. Louis, MO, USA) were used. After binding to the indicated secondary antibodies, the detection of the antigen–antibody complex was performed using an enhanced chemiluminescence (ECL) blotting analysis system (GE Healthcare Life Sciences, Piscataway, NJ). The band of Western blot analysis was then quantified using ImageJ software (v1.44m for Windows, National Institutes of Health, Bethesda, MD, USA).