Anti rarβ

Total protein was isolated from 80 to 90 % confluent cell cultures using RIPA buffer, total concentration of extracted proteins was determined by Bradford assay. Total protein concentration of each sample was adjusted to 1 μg/μL. Proteins were separated based on their weight and electrophoresis charge in 10 % acrylamide gels. Samples were placed in wells and electrophoresed at 180 V and 300 mAmps for one hour. Proteins were then transferred to nitrocellulose membranes (0.45 μm) at 100 V and 300 mAmps for 1 h. Membranes were then blocked with 5% skim milk in TBS 1x for 1 h in agitation and then incubated overnight at 4 °C with corresponding primary antibody Anti-TLR3 (1:2000), Anti-RARβ (1:2000) or Anti-β-actin (1:2000) (Abcam, Cambridge, CB2 0AX, UK). HRP-labeled secondary antibody (Abcam) was added to membranes and incubated for 2 h at room temperature. Bands corresponding to proteins of interest were visualized by incubating membranes with chemiluminescent HRP substrate (Luminol^®) followed by exposure to a photographic film. β-Actin expression was used as loading control.