Hybond p polyvinylidene difluoride membrane

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.

HEC-1B and Ishikawa protein was extracted using the RIPA Lysis Buffer System (Santa Cruz Biotechnology, Dallas, TX, USA). Five micrograms of protein (whole cell extracts) was subjected to sodium dodecyl sulfate-poly acrylamide gel electrophoresis (10% acrylamide gel). Following SDS-PAGE, proteins were transferred onto Hybond-P Polyvinylidene Difluoride membrane (GE Healthcare, Little Chalfont, UK). Anti-phospho-β-catenin antibody (#4176) was purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin was purchased from Sigma, and anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-Horseradish peroxidase was purchased from Wako. Antibody–protein complexes were detected on the blots using ECL Plus Western blotting detection reagent (GE Healthcare), and the protein bands were visualized with a LAS-4000 image analyzer (Fuji Photo Film, Tokyo, Japan). The relative intensity values of the protein bands were measured by the use of an imaging analyzer, Lumina Vision (Mitani Corp., Fukui, Japan). The expression level for phospho-β-catenin was summarized as a ratio of GAPDH.

Protein was extracted from cultured cells using Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL, USA) added with Halt Protease Inhibitor Cocktail (Pierce Biotechnology). The protein extracted from whole cell (20 μg) was analyzed by SDS-PAGE (10% acrylamide gel) and transferred onto Hybond P polyvinylidene difluoride membrane (GE Healthcare, Little Chalfont, UK). Primary antibodies employed in this study were anti-human 17β-HSD2 (10978-1-AP; Proteintech, Chicago, IL, USA) and anti-human β-actin (AC-15; Sigma-Aldrich). Antibody–protein complexes were detected using ECL plus Western Blotting Detection reagents (GE Healthcare), and visualized with LAS-1000 image analyzer (Fuji Photo Film, Tokyo, Japan). The intensity of each band of 17β-HSD2 was quantitated by using the Imaging Analyzer (Lumina Vision; MITANI CORPORATION, Tokyo, Japan). The data were demonstrated as an intensity score in Figure 2.

Stimulated HBSMCs were harvested with a cell scraper and were solubilized in a lysis buffer consisting of 20 mM Tris–HCl (pH 7.5), 1 % Nonidet P-40, 1 mM EDTA, 50 mM NaF, 50 mM sodium β-glycerophosphate, 0.05 mM Na₃VO₄, 10 μg/ml leupeptin, and 100 μM phenylmethylsulfonyl fluoride. Following centrifugation at 5000g for 5 min, the resultant supernatant was used as the lysate after protein concentration determination using the Bradford assay (Bio-Rad, Hercules, USA). The lysates were resolved in a 10 % SDS polyacrylamide gel and were electrotransferred to Hybond-P Polyvinylidene difluoride membrane (GE healthcare, Amersham Place, UK). Immunodetection of JNK1 and phosphorylated JNK1 was performed using anti-JNK1 (1:1000 dilution; Cell Signaling, Massachusetts, USA), and anti-phosphorylated JNK1 (Thr183/Tyr185) antibodies (1:1000; Cell Signaling), respectively. The signal was detected by incubation with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10,000; Promega, Tokyo, Japan), followed by chemiluminescence detection using the SuperSignal kit (Pierce Chemical Company, Rockford, USA) and Kodak BioMax light film (Kodak, Tokyo, Japan). The densities of the bands were quantified using the computer software, ImageJ (developed at the U.S. National Institutes of Health).

Protein expression was assessed by Western blot analysis as described previously [4 (link)]. Briefly, cells treated with the indicated reagents were washed in ice-cold phosphate-buffered saline (PBS) and lysed in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea). Aliquots of cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto a Hybond-P⁺ polyvinylidene difluoride membrane (GE Healthcare, Little Chalfont, UK). Membranes were blocked overnight at 4°C with 5% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20. After a brief wash with TBS, the membranes were incubated overnight at 4°C with the indicated primary antibodies in TBS containing 0.05% Tween-20, followed by peroxidase-conjugated mouse, goat, or rabbit antibodies for 1 h at room temperature. After extensive washing in TBS containing 0.1% Tween-20, immunoreactive bands were detected using West-ZOL Plus (iNtRON Biotechnology).

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.

Transfected DLD1 or HeLa cells were used for co-immunoprecipitations as describe previously [27 (link)] using 20 µl Flag M2 monoclonal antibody affinity gel (Sigma–Aldrich, A2220). For the precipitation of endogenous GBP-1, beads were prepared by covalent cross-linking of the α-GBP-1 antibody (1B1) [7 (link)] as described before [28 (link)].
Precipitated proteins and cell lysates were separated under reducing conditions in 10–15% sodium dodecyl sulfate–polyacrylamide gels and transferred onto a Hybond-P polyvinylidene difluoride membrane (GE Healthcare). Membranes were blocked for 1 h with 5% skim milk in 1× PBS and incubated overnight (4°C) with the following primary antibodies: mouse-α-Flag M2 (Sigma, F3165, 1 : 10 000), rabbit-α-β-actin (Abcam, ab8227, 1 : 5000), rabbit-α-panTEAD (Abcam, ab197589, 1 : 1000), rabbit-α-TEAD1 (Abcam, ab133535, 1 : 1000), rabbit-α-TEAD2 (Avia Systems Biology, OAAB18572, 1 : 1000) or rabbit-α-TEAD3 (Santa Cruz, L-17, sc-102130, 1 : 500), and rat-α-GBP-1 (1B1 [7 (link),28 (link)], hybridoma supernatant, 1 : 500). The respective secondary antibodies (Dako, 1 : 5000) were coupled to horseradish peroxidase (incubation for 1 h). Signals were detected with the ECL Prime Western Blotting System and the Amersham Imager 600 (both GE Healthcare). Quantification was performed using the ImageQuantTL software (GE Healthcare).