Ecl plus blotting detection system

Forty-eight hours post transfection cellular lysates or cellular medium were collected for experiments. Cells were lysed in equal volumes of pre-heated 2xSDS loading buffer (Sodium Dodecyl Sulphate 125 mM, Tris-HCl pH 6.8, 20% Glycerol, 2% SDS, 2% β-mercaptoethanol and bromophenol blue) and homogenized via sonication. Antibodies used against the ER-Stress proteins were: anti-BiP, anti-PERK, anti-P-PERK (Cell Signaling Technology, Danvers, MA) and anti-CHOP, anti-p-eIF2a (SantaCruz Biotechnology, CA), followed by peroxidase-labelled secondary antibodies either goat anti-mouse or donkey anti-rabbit (SantaCruz Biotechnology, CA). Proteins were detected using the Enhanced ChemiLuminescence (ECL) Plus Blotting Detection system (Amersham Biosciences, Buckinghamshire, UK) and were visualized by autoradiography on photographic film (KODAK X-OMAT, NY). All transblots were reprobed with anti-β-tubulin antibody (SantaCruz Biotechnology, CA) to prove equal amounts of protein were loaded on the membrane. Band density was defined using the ImageJ Software (http://imagej.nih.gov/ij).

AB8/13 cells were lysed in equal volumes of pre-heated 2xSDS loading buffer (Sodium Dodecyl Sulphate–125 mM Tris-HCl pH 6.8, 20% Glycerol, 2% SDS, 2% β-mercaptoethanol and bromophenol blue) and homogenized using a 2 ml syringe. Whole cell lysates were subsequently electrophoresed in a 12% SDS-Polyacrylamide gel. Gel transfer was held in a wet transfer system on Hybond Polyvinylidene Fluoride (PVDF–Millipore, MA) membranes. Membranes were blocked with 5% non-fat dry milk in PBS/0.01% Tween20 for 1 hour at room temperature. Primary antibody was diluted in milk and added to the membrane for one hour. TFCP2L1 protein was detected with the goat primary polyclonal antibody C-20 (SantaCruz Biotechnology, CA) at around 48 kDa. β-Tubulin was used as loading control by using the T-4026 primary antibody (Sigma, Taufkirchen, Germany). As secondary antibody we used the rabbit anti-mouse antibody (SantaCruz Biotechnology, CA), conjugated with Horseradish Peroxidase (HRP). Proteins were detected using the Enhanced ChemiLuminescence (ECL) Plus Blotting Detection system (Amersham Biosciences, Buckinghamshire, UK) and were visualized on a ChemiDoc MP imaging system using the Image Lab software (Bio-Rad Laboratories).