Mouse anti iκb

Protein was extracted from the 5 samples from each group, electrophoresed with 4-20% sodium dodecyl sulfate (SDS) -polyacrylamide gradient gel, and transferred onto polyvinylidene fluoride transfer membranes (Immobilon-P, 0.45 μm, Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies: rabbit anti-COX2 (1:200, ab15191, Abcam, Cambridge, MA, USA), mouse anti-GFAP (1:1000, MAB360, Millipore), rabbit anti-iNOS (1:200, ab15353, Abcam), mouse anti-MAP-2 (1:1000, MAB3418, Millipore), rabbit anti-NF-κB p65(1:1000, #8242, Cell Signaling, Danvers, MA, USA), mouse anti-Phospho-NF-κB p65 (1:1000, #3036, Cell Signaling), mouse anti-IκB (1:1000, #9242, Cell Signaling), mouse anti-Phospho-IκB (1:1000, #9246, Cell Signaling), and rabbit anti-TLR-4 (1:200, sc16240, Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with peroxidase-conjugated secondary antibodies. The signals were detected by an enhanced chemiluminescence system (E2400, Denville Scientific, South Plainfield, NJ, USA). The same membranes were incubated with antibody for β-actin as a control after washing with stripping buffer (21059, Thermo Scientific, Waltham, MA, USA). The blotting signals were quantified using ImageJ software (NIH) and presented as adjusted density normalised to density of actin in the same gel.