Western Blotting
Electrophoresis was performed using 10% polyacrylamide gel and the Bio-Rad mini-Protean III apparatus (Bio-Rad, Hercules, CA, U.S.A.). In brief, the proteins present in cytosolic and nuclear fractions were size-separated in 10% SDS-PAGE (90 V). The immunoblotting was performed as described previously (Kawamoto, Lepsch et al., 2008)The proteins were blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, U.S.A.) and incubated with the specific antibody: RelA (p65) (1:1000, sc-0372; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (1:2000 (58169; Cell Signaling, U.S.A.) and after with secondary antibody (Rabbit). Proteins recognized by antibodies were revealed by an electrochemiluminescence (ECL) technique, following the Manufacturer’s instructions (Amersham Biosciences, Amersham, U.K.). To standardize and quantify the immunoblots, we used the photo documentation system DP-001-FDC (VilberLourmat, Torcy, France) and N.I.H. ImageJ software (http://rsb.info.nih.gov/ij). Several exposure times were analyzed to ensure the linearity of the band intensities.