Western Blot Analysis of Protein Expression

Proteins were extracted in RIPA buffer and were separated (30–100μg per track) by SDS-PAGE (7.5%, 10% or 15%); blotting and washing were performed as previously described [30 (link)]. The blots were incubated in 5% non-fat milk PBS 0.1% (v/v) Tween 20 with the appropriate antisera. Antibodies (with dilutions) used were directed to: LMP1 (IG6) 1:100, TGFα (Santacruz) 1:1000, Chil3/4(YM1/2) (R&D systems) 1:1000, SOD1 (Santacruz) 1:200, Erk1/2 (Cell Signaling) 1:1000, phosphor-Erk1/2 (Cell Signaling) 1:1000, Stat3 (Cell Signaling) 1:1000, S100A9 (R&D Systems) 1:1000, EGFR (Cell Signaling) 1:1000, IκBα (Cell Signaling) 1:1000, phosphoIκBα (Cell Signaling) 1:1000, GAPDH (Santacruz) 1:1000, actin (Santacruz) 1:1000, IgE (Abcam) 1:1000, followed by the appropriate 1:4000 goat anti-mouse, anti-rabbit, anti-rat, or donkey anti-goat IgG HRP-conjugates (Santacruz). Mouse IgM, IgA and IgG were detected directly using antibody HRP-conjugates (Santacruz or Southern Biotech.) 1:4000. Detection was performed by enhanced chemiluminescence (liteAblot kit, Euroclone) and bands on blot images quantified using imageJ.