C abl

Cells or brain tissues were lysed in 300 μl of lysis buffer [150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 50 mM Tris-HCl (pH 7.5), 2 mM EDTA] containing mixture of Halt^TM protease and phosphatase inhibitors (1×) (Thermo Fisher Scientific). The brain tissues were individually homogenized and then centrifuged at 13,400×g at 4°C for 15 min. Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Bovine serum albumin was used as the standard. Proteins (20–30 μg) for each sample were separated using 12% sodium dodecyl sulfate-PAGE and transferred to polyvinylidene fluoride filter membranes (Bio-Rad) by the semi-dry electroblotting method. The membranes were blocked with 5% skim milk and incubated sequentially with the following primary antibodies against either c-Abl (rabbit monoclonal antibody, 1:1000; Santa Cruz), p-p65, p-65, p-IκB, IκB (rabbit monoclonal antibody, 1:1000; Cell Signaling), TNF-α (rat anti-mouse monoclonal antibody, 1:500; Millipore) or α-tubulin (mouse monoclonal antibody, 1:2000; Sigma-Aldrich) and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or mouse IgG antibody; Cell Signaling), followed by chemiluminescence detection (Thermo Fisher Scientific).