Hybond p pvdf membrane

The Lowry method was used to evaluate the protein content after the cells had been seeded in 6-well plates, cultured in complete media, and lysed using ice-cold lysis buffer supplemented with protease inhibitor cocktails (Roche Molecular Biochemicals, Mannheim, Germany) (Biorad DC Protein Assay, MA, USA). In the running buffer, 25 µg of protein from each sample was placed onto a polyacrylamide gel and electrophoretically separated. The proteins were blotted onto a Hybond-P PVDF membrane following electrophoresis (Amersham Biosciences, Buckinghamshire, UK). The membrane was exposed to anti-TOM20 (mouse, 1:1000; AbCam, Cambridge, UK), anti-VDAC1 (mouse, 1:1000; AbCam, Cambridge, UK), and anti-BNIP3 (rabbit, 1:1000; AbCam, Cambridge, UK) after blocking with a 10% skim milk solution. Following washing, the membrane was incubated with HRP-conjugated anti-rabbit secondary antibody (1:3500; PerkinElmer, MA, SUA) or anti-mouse secondary antibody (1:10000; PerkinElmer, MA, USA). According to the manufacturer’s instructions, the signal was seen using an improved chemiluminescent kit from Amersham Biosciences, and then it was examined using a Molecular Imager VersaDoc MP 4000 (Biorad, Hercules, CA, USA). Proteins were normalized to β-ACTIN (mouse, 1:7000, AbCam, Cambridge, UK) and GAPDH (rabbit, 1:2000, Cell Signaling, Danvers, MA, USA).

Cells were lysed in a lysis buffer containing (50 mM Tris, 25 mM β-glycerophosphate, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% (v/v) Triton X100, 1 mM DTT, 1 mM benzamidine, protease and phosphatase inhibitor cocktail (Sigma). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a Hybond-P PVDF membrane (Amersham Biosciences). Membranes were blocked for 1 h at room temperature with a solution of 5% nonfat milk powder in TN (50mM Tris-HCL, pH 8.0, 150mM NaCl, 0.1% Tween-20). The blots were then incubated with antibody in blocking solution overnight at 4°C. Antibodies were diluted according to the manufacturer's instructions. The blots were washed three times in TN and incubated with horseradish peroxidase conjugated goat anti-IgG (1:5000) (Santa Cruz, CA) in blocking buffer for 1 h at room temperature. After three washes, the blots were developed using the LuminataTM Western HRP substrate (Millipore Billerica, MA).

After electrophoretic separation by SDS-PAGE, proteins were transferred on to a Hybond-P PVDF membrane (Amersham - GE Healthcare). PVDF membranes were incubated in blocking solution (5% milk, 50 mM Tris HCl pH 7.0, 0.05% Tween-20) for 1 h and probed overnight with mouse Anti-GPR54 (1∶500 dilution), rabbit Anti-Kisspeptin (1∶100 dilution), rabbit Anti-ERK1/2 and Anti-P-ERK1/2 (1∶1000 dilution). Then Anti-Mouse or Anti-Rabbit HRP-conjugated secondary antibodies (1∶5000 dilution) were added followed by addition of SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and quantified using the BioSpectrum 500 Imaging System (UVP).

The cells were harvested, resuspended in PBS, 0.5% Triton X 100 (v/v) (Sigma), 0.02% (w/v) sodium azide (Sigma), and 2 mM phenylmethylsulfonyl fluoride (PMSF, Cell Signal) and incubated for 10 min on ice. The nuclei were then pelleted by centrifugation at 4°C and resuspended in 20 mM HEPES‐KOH (Sigma), 420 mM NaCl (Sigma), 25% (v/v) glycerol (Sigma), 1.5 mM MgCl₂ (Sigma), 0.2 mM EDTA (Sigma), 0.5 mM dithiothreitol (Sigma), 0.2 mM PMSF, supplemented with protease inhibitor cocktail (Sigma) for 20 min on ice. The lysate was centrifuged at 4°C and the supernatant was used in all Western blot analyses.
Western blot was performed on 8% SDS‐polyacrylamide (Bio‐Rad) resolving gels using 80 µg of protein per lane and the separated protein was transferred to a hydrated HYBOND–P PVDF membrane (Amersham). Immunoblotting with primary antibody was carried out at 4°C overnight using a 1:1000 dilution of rabbit anti‐REST (OriGene, TA330562) and a 1:10000 dilution of mouse anti‐β‐actin (Sigma, A1978) antibodies. The detection with secondary HRP‐conjugated antibodies was performed using a dilution of 1:20000 anti‐rabbit (Cell Signalling) and 1:40000 dilution of anti‐mouse (Cell Signalling) antibodies for 1 h at room temperature. Immunoreactive bands were visualized by ECL kit (Enhanced Chemiluminescence, GE Healthcare) and scanned using the LAS‐3000 intelligent dark box (Fujifilm).

The three hiPSC lines were incubated with or without BMP4 (50 ng/mL) for 4 hours. Protein was extracted as previously described [16 (link)] and concentration was determined by BCA assay (Pierce). Fifteen μg of protein was separated by SDS- PAGE (Invitrogen) and transferred to Hybond-P PVDF membrane (Amersham). The membrane was blocked with 5% non-fat dried milk in TBST for 30 minutes, incubated with diluted PhosphoSmad1/Smad5/Smad8 antibody (9511S, Cell Signaling Technology) in 5%(w/v) BSA, 1xTBST at 4°C with gentle shaking overnight, then incubated with HRP-conjugated donkey anti-rabbit IgG (1:2,000 in TBST, Cell Signaling Technology) at ambient temperature for 1 hour. Reactive protein bands were visualized using ECL plus Western blotting detection reagents (GE Healthcare) and band intensities were calculated using the Bio Rad Chemi Doc XRS imaging system. Beta-actin (13E5) Rabbit mAB (4970, Cell Signaling Technology) was used for a loading control.

NCTD (99.5%), collagen, luciferin–luciferase, U46619, heparin, prostaglandin E₁ (PGE₁), bovine serum albumin (BSA), arachidonic acid (AA), fibrinogen, FITC‐phalloidin and thrombin were purchased from Sigma‐Aldrich (St. Louis, MO, USA). An anti‐integrin β₃ monoclonal antibody (mAb) and anti‐phospho‐integrin β₃ (Tyr⁷⁵⁹) polyclonal antibody (pAb) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti‐phospho‐Src family (Tyr⁴¹⁶) and anti‐phospho‐FAK (Tyr³⁹⁷) mAbs, as well as an anti‐Src family pAb, were purchased from Cell Signaling (Beverly, MA, USA). An anti‐Focal adhesion FAK pAb was obtained from Millipore (Billerica, MA, USA). FITC‐anti‐human CD42P (P‐selectin) and FITC‐anti‐human CD41/CD61 (PAC‐1) mAbs were obtained from BioLegend (San Diego, CA, USA). Protein G Mag Sepharose Xtra Beads were purchased from GE Healthcare (Uppsala, Sweden). A Hybond‐P PVDF membrane, an enhanced chemiluminescence Western blotting detection reagent, horseradish peroxidase (HRP)‐linked donkey antirabbit immunoglobulin G (IgG) and sheep antimouse IgG were purchased from Amersham (Buckinghamshire, UK). The Dade Behring PFA‐100 collagen/ADP (CADP) test cartridge was obtained from Siemens Healthcare (Erlangen, Germany).