Species specific horseradish peroxidase conjugated secondary antibodies

CVPs were cultured in basal medium in the presence or absence of endothelin-1 growth factor for 5 days. For the preparation of cell lysates, cell pellets were incubated in RIPA lysis buffer (1% Nonidet P-40, 0.5% deoxycholate, 5 M NaCl and 1 M Tris (pH 7.4)) for 10 min at 4 °C, and then centrifuged at 11,400g for 15 min at 4 °C. Quantification of protein extract was carried out using the Protein Assay (Bio-Rad) according to the manufacturer's instructions. Electrophoretic analysis was performed using 10–15% SDS–PAGE gel (Bio-Rad). Gels were blotted onto nitrocellulose membrane (Amersham Biosciences), which were then probed with rabbit anti-human AKT, anti Phospho-AKT (Ser473; Cell Signaling), mouse anti β-actin (Santa Cruz), rabbit anti-human HEY1 and HEY2 (both from GeneTex) and mouse anti α-tubulin (Santa Cruz) primary antibodies according to the manufacturer's instructions. Primary antibodies were detected with species-specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) and ECL western blotting detection system (Amersham Biosciences).

Cells were lysed by agitation on a rotating platform at 4°C for 1 hour in lysis buffer containing 50 mM Tris/ HCl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride , and 10 mg/mL aprotinin. Cellular debris was removed by centrifugation at 16,060 × g and 4°C for 20 min. An equal amount of protein of each cell extracts was subjected to 10% SDS-PAGE and the proteins were transferred using semi-dry blotting to Hybond-ECL membranes (GE Healthcare, Glattbrugg, Switzerland). Western blot analysis was performed by probing the membrane with the following antibodies at the indicated dilution as follows: anti-rabbit ΔNp63 (1:500; provided by Dr. James DiRenzo, Dartmouth Medical School), anti- goat GLI2 (1:200, Santa Cruz Biotechnology, CA, USA), anti-rabbit GAPDH (1:3000; Santa Cruz Biotechnology) and species specific horseradish peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology). Peroxidase activity was detected using the Immobilon chemiluminescence substrate (Millipore, Billerica, MA, USA) and the signals were recorded using a VersaDoc Imaging System (Bio-Rad, Hercules, CA, USA).

hCMs were treated with IFN-γ alone or IFN-γ + 1-MT for increasing amounts of time. hCMs were lysed with 1X Laemmli sample buffer (62.5 mM Tris–HCl [pH 6.8], 10% glycerol, 1% sodium dodecyl sulfate [SDS], and 5% β-mercaptoethanol) and boiled for 5 min. Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA, USA). Then, the membrane was blocked for 30 min in Tris-buffered saline containing 5% non-fat and 0.05% Tween-20, followed by incubation with a primary antibody against IDO1, IDO2, IRF-1, Fas, Fas ligand (FasL), glyceraldehyde 3-phosphate dehydrogenase (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cellular (ED-A) fibronectin (Sigma), or α-smooth muscle actin (SMA) (Abcam, Cambridge, MA, USA). Species-specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) were used to detect bound primary antibodies. Visualization of protein bands was performed with the West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA) and the BioSpectrum imaging system (UVP, Upland, CA, USA).