Ab32518

Immunoblotting analysis was conducted as previously described.17 (link) The antibodies used in these analyses included anti-RIP1 (ab72139, 1:1,000; Abcam, Cambridge, UK), anti-IκBα (ab32518, 1:1,000; Abcam), anti-phospho-IκBα (ab133462, 1:10,000; Abcam), anti-VEGF-C (ab9546, 1:1,000; Abcam), anti-TAK1 (sc-7967, 1:1,000; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-NEMO (ab97642, Abcam, 1:1,000), and anti-GAPDH (ab8245, 1:1,000; Abcam). The detected proteins were semiquantified relative to GAPDH expression in each gel.

Hippocampal tissue samples were collected, and protein lysis solution (RIPA) was used for lysing tissues on ice for 30 minutes to extract proteins. After protein quantification, 10% SDS-PAGE was performed, and the protein on the gel was transferred to a PVDF membrane by the wet transfer method. After blocking the PVDF membrane with blocking solution for 2 hours at room temperature, rabbit monoclonal antibody BDNF (1:1,000, ab108319), rabbit monoclonal antibody TrkB (1:1,500, ab18741), rabbit monoclonal antibody p-IKKβ (1:1,000, ab38515), rabbit monoclonal antibody IKKβ (1:1,000, ab124957), rabbit monoclonal antibody IκBα (1:1,000, ab32518), rabbit polyclonal antibody NF-κB p65 (1:1,500, ab16502), and rabbit polyclonal antibody GAPDH (1:1,000, ab9485, Abcam, Cambridge, UK) were applied and incubated overnight at 4 °C. The next day, membranes were washed with PBST for 30 minutes, then incubated with an HRP-labeled secondary antibody at 37 °C for 1 hour, and the membrane was washed with PBST for 1 hour. Finally, the target protein was detected using the enhanced chemiluminescence ECL kit, and the gray value was analyzed by Image J software.

The brain tissues were added with pre-cooled Tris-HCl buffer (pH 7.4) at the ratio of 1:3, and made into brain tissue homogenate with a glass homogenizer in an ice bath, and then the cells or tissue homogenates were lysed in cold RIPA containing a protease inhibitor cocktail [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM PMSF, 1 mM NaF, 1 mM Na₃VO₄, 1% protease inhibitor cocktail from Sigma, which was added right before use], for 30 min. The lysate was then centrifuged at 4°C for 20 min at 16,000 × g to collect the supernatant. The protein concentration was determined using the Pierce bicinchoninic acid assay kit (Beyotime Biotechnology Co., Ltd.). Equal amounts of protein (20 μ.l/well) were separated by 7.5% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for 1 h and incubated overnight at 4°C with primary antibodies as follows: Recombinant nuclear factor of activated T cells c2 (NFATc2) (1:1,000, ab2722; Abcam), p65 (1:1,000, ab16502; Abcam), p-p65 (1:2,000, ab86299; Abcam), IκBα (1:1,000, ab32518; Abcam), and p-IκBα (1:10,000, ab133462; Abcam). The membranes were then incubated with secondary antibody IgG (1:2,000, ab205718; Abcam). The gray value of the target band was analyzed using ImageJ software (Rawak Software, Inc.) with β-actin as the internal reference.