Nonfat dry skim milk powder

Total protein was extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) from cultured cells. Following measurement of protein concentration (Protein Assay Rapid Kit Wako; Wako Pure Chemical Industries, Osaka, Japan), the total protein was individually subjected to SDS-PAGE (SuperSep Ace; Wako, Japan). These proteins were transferred onto Hybond P polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK). Then the proteins on the membrane were blocked in 5% nonfat dry skim milk powder (Wako) for over 1 h at room temperature and were incubated with primary antibodies for 24 h at 4° C using Immuno Shot (Cosmo Bio, Tokyo, Japan). The dilutions of primary antibodies used in this study were as follows: p22^phox, 1:100;HIF-1α,1:200; E-Cadherin, 1:200; Vimentin, 1:200; β-actin, 1:1000. These antibody–protein complexes on the blot were detected using ECL-plus Western blotting detection reagents (GE Healthcare) following incubation with anti-rabbit or anti-mouse IgG horseradish peroxidase (GE Healthcare) at room temperature.

Total protein was extracted using the PhosphoSafe Extraction Reagent (Biosciences Inc., Darmstadt, Germany) from cultured cells. Following the measurement of protein concentrations (Protein Assay Rapid Kit Wako, Wako), the total proteins were individually subjected to SDS-PAGE (SuperSep Ace, Wako). These proteins were transferred onto the Hybond P polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK). Next, the proteins on the membrane were blocked in 5% non-fat dry skim milk powder (Wako) for over 1 h at room temperature and were incubated with primary antibodies overnight at 4 °C using ImmunoShot (Cosmo Bio Co., Ltd., Tokyo, Japan). The dilution of primary antibodies used in this study was as follows: 11βHSD1, 1:500; 11βHSD2, 1:1000; GR, 1:1000; β-actin (Sigma-Aldrich Co., St. Louis, MO, USA), 1:1000. These antibody–protein complexes were detected on the blot using ECL-plus western blotting detection reagents (GE Healthcare) following an incubation with anti-rabbit or anti-mouse IgG horseradish peroxidase (GE Healthcare) at room temperature for 1 h.

Total protein was extracted using PhosphoSafe Extraction Reagent (Biosciences, Darmstadt, Germany) from cultured cells. Following measurement of protein concentration (Protein Assay Rapid Kit Wako; Wako Pure Chemical Industries, Osaka, Japan), the total protein was individually subjected to SDS-PAGE (SuperSep Ace; Wako, Japan). These proteins were transferred onto Hybond P polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK). Then the proteins on the membrane were blocked in 5% nonfat dry skim milk powder (Wako) for over 1 h at room temperature and were incubated with primary antibodies for 24 h at 4 °C using Immuno Shot (Cosmo Bio, Tokyo, Japan). The dilutions of primary antibodies used in this study were as follows: MMP-1, 1:2000; EGFR, 1:1000; pEGFR, 1:500; mTOR, 1:1000; pmTOR, 1:500; β-actin, 1:1000. These antibody–protein complexes on the blot were detected using ECL-plus Western blotting detection reagents (GE Healthcare) following incubation with anti-rabbit or anti-mouse IgG horseradish peroxidase (GE Healthcare) at room temperature.