Then the pellet cells were placed in the SORVALL RC-5 (8000 rpm, 4°C, 10 min) and solubilized in buffer B containing 1% DM and 0.1 mM PMSF for 1 h at 4°C.
Cells were resuspended in cold buffer W (50 mM Tris pH 8.0, 250 mM NaCl, 5 mM EDTA, 1 mM PMSF, 10 µg/mL leupeptin, 0.1 mM aprotinin,10 µg/mL DNase 1, 10 µg/mL lysozyme), sonicated and inclusion bodies collected by centrifugation at 30,000×g, 20 min and solubilized in buffer G (6 M GdmCl, 20 mM Tris pH 8.0, 50 mM Na2HPO4, 100 mM NaCl, 10 mM reduced glutathione, 10 mM imidazole).
The paraformaldehyde-fixed sections were treated with 50 mM NH4Cl in PBS for 20 min and solubilized with 0.1% Triton X-100 for 5 min.
The pellets were collected by centrifugation at 15000 × g for 1 min and solubilized in 200 μL of denaturing lysis buffer with 1% SDS without dithiothreitol prepared as described by Bonifacino et al. [ 21].
The harvested cells were resuspended in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM dithiothreitol (DTT), lysed (M-110L Microfluidizer Processor, Microfluidics, Newton, MA, USA), pelleted (20,000 g, 4°C, 20 min) and solubilized in the presence of 1% n-lauroylsarcosine (N-LS) for 1 h at 4°C.
Lysates were centrifuged at 5,000×g for 20 min, and solubilized proteins were precipitated with the indicated antibodies, separated by SDS-PAGE, and revealed by western blot with the anti-Akt substrate antibody that recognizes all the phosphorylated Akt substrates (Cell Signaling, Danvers, MA USA).
For quantitation of total adherent cell number, cells were fixed with 1% gluteraldehyde (Fisher Scientific, Pittsburgh, PA) for 15 min, incubated with 0.1% crystal violet (Fisher Scientific) for 30 min, destained with H2O, and solubilized with 0.2% Triton X-100 (Sigma).
B16F10 and HaCaT cells were cultured in HBSS containing 1.0 mM RD, RK, MEHQ, pHPA, 2pHP-IA, or pHPE and 20.0 μM DCFDA for 30 min, washed with HBSS, and solubilized with 0.5% Triton X-100 reduced.
After the addition of 1 ml TCA, the cells were placed at 4°C for 30 min, washed twice with PBS, and solubilized with 500 µl 0.5 M NaOH, 0.5% SDS.
H9 cells treated with BMP-2, BMP-6 or BMP-2/6 at 100 ng/ml for 5, 10, 30, 60 or 120 min were washed with PBS, and solubilized in RIPA buffer containing 50 mM Tris-HCl pH7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate.
After centrifugation at 16,000 r.c.f. for 15 min, the pellets were dried and solubilized in buffered urea (6 M urea, 2 M thiourea, 100 mM Tris-Cl at pH 8.0, 20 mM DTT).
