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.