INTRODUCCIÓN: REVISIÓN CRITICA DEL PROBLEMA

ANEXOS
buffer (Buffer A) composed by (in mmol · L -1 ): 10 HEPES, 1 EDTA, 1 EGTA, 10
KCl, 1 dithiothreitol (DTT) and antiproteases (Roche, Mannheim, Germany) at pH
of 7.9. Cells suspension was then lysed by adding NP-40 0.5% (v/v) to the
reaction and incubating during 10 min. Cell lysate obtained was vortexed during 15
seconds and centrifuged (200 x g, 5 min, 4 ºC) to separate non-lysated cells. The
supernatant was recovered to another tube and centrifuged (1500 x g, 15 min, 4
ºC) to separate the cytoplasm proteins (in the supernatant) and the nucleus (in the
pellet). Nucleus were then lysed by incubation in a solution of Buffer A
supplemented with glycerol 20% (v/v) and KCl 0.4 mol · L -1 during 30 minutes with
periodical vortexing. Nuclear proteins were recovered from the supernatant after a
final centrifugation (13000 x g, 15 min, 4 ºC).
d) Western blot
Western blot analyses were performed on nucleus and cytoplasm proteins
extracts. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) in 10% (w/v) polyacrylamide gels and transferred to
a polyvinylidene difluoride (PVDF) membrane over 45 min at 280 mA. The PVDF
transferred membranes were blocked for 1 hour at room temperature with
skimmed milk 5% (w/v) in Tris-buffered saline Tween-20 (TBST) solution
containing 20 mmol · L -1 Tris-HCl (pH 7,6), 150 mmol · L -1 NaCl, and 0.1% (v/v)
Tween 20. Membranes were then incubated with rabbit antibodies for p65 subunit
of NF-κB (1:500 dilution; SantaCruz Biotechnology, California, USA) overnight at 4
ºC. After washing with TBST, membranes were incubated with peroxidase-
conjugated goat anti-rabbit IgG (1:2,000 dilution; 1h at room temperature; Fisher
Scientific, Madrid, Spain). Immunoreactive proteins were visualized using an
enhanced chemiluminescence detection system (Fisher Scientific, Madrid, Spain),
and were quantified densitometrically using ImageJ (1.37v) software (U.S.
National Institutes of Health, Bethesda, Maryland, USA). Results were expressed
as the nucleus/cytoplasm ratio of the optical densitometry for p65 subunit, with
respect to HSA treatment. Possible contamination of nucleus extract with
cytoplasm proteins was checked by incubation with anti-human glyceraldehyde 3-
phosphate dehydrogenase antibody (GAPDH; 1:1,000 dilution; SantaCruz
Biotechnology, California, USA).
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