Ac 15

Hippocampus and cortex were washed in ice-cold PBS and lysed by sonication with RIPA-DOC buffer containing 50 mM TrisHCl (pH 7.2), 150 mMNaCl, 1% deoxycholate, 2–3% Triton X-100, 0.1% SDS and protease inhibitor cocktail Complete (Roche). Protein lysates were diluted in 4XSDS-sample buffer and separated on 12% Tris-glycine SDS-PAGE geland transferred to polyvindylidine fluoride (PVDF-FL) membranes. Membranes were blocked in PBS containing 5% non-fat dried milk and incubated with the primary antibodies diluted in the blocking buffer at 4 °C overnight. Rabbit anti-TMP21 antibody T21 (1:1000) was generated by inoculating rabbit with synthetic peptide HKDLLVTGAYEIHK, this peptide shared 100% sequence homology with both mouse and human TMP21 [30 (link)]. Human p24a was detected by mouse monoclonal antibody TMED2 (1:2000) (C-8) (Santa Cruz Biotechnology). The antibody AC-15 (1:5000) (Abcam, Cambridge, MA, USA and Sigma) was used to detect β-actin. Then the membranes were rinsed in PBS-T and incubated with near-infrared fluorescence-labeled secondary antibodies IRDyeTM680-labeled goat anti-rabbit (1:100,000) and IRDyeTM800-labeled goat anti-mouse antibodies (1:100,000)(Lincoln, NE, USA)in PBS-T at room temperature for 1 h, after further rinsed in PBS-T the membrane was scanned by LI-COR Odyssey R system.

Cellular extracts from cell lines were prepared by dissociation in lysis buffer, and protein was measured using a BCA protein assay kit. Proteins were resolved by SDS-PAGE using the laemmli buffering system with 15% polyacrylamide running gels and 5% stacking gels, and then transferred to reinforced PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 3% skim milk for 1 hour, the membranes were incubated with primary antibodies against leptin-receptor (sc-8391, 1:500, Santa Cruz,) and caspase-3 (H-227) (sc-7148, 1:1,000, Santa Cruz) for 2 hours at room temperature. After washing, the blots were incubated for 45 minutes at room temperature with a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (Abcam, Cambridge, UK). After extensive washing, the antigen-antibody complexes were visualized by exposing the membranes to X-ray film (Fuji, Japan). An antibody against beta-actin antibody (AC -15, 1:10000, Abcam, Cambridge, UK) was used to verify equal loading and transfer of total proteins.

Total protein in tissues and cells was extracted with RIPA lysis buffer (R0010, Solarbio) containing PMSF. The cells were incubated for 30 min on ice, centrifuged at 12000 r/min, and 4°C for 10 min, and the supernatant was taken. The BCA kit (23225, Pierce) was employed to determine protein concentration of each sample. A 10% SDS-PAGE (P0012A, Beyotime Institute of Biotechnology, Shanghai, China) was adopted to dissolve proteins, which were transferred to a PVDF membrane (ISEQ00010, Millipore, Billerica, MA, USA) by wet transfer. The PVDF membrane was blocked with TBST buffer containing 5% skimmed milk powder for 2 h. The blots were probed with the primary antibody to rabbit KDM6A (1 : 1000, ab36938, Abcam, UK), NOTCH2 (1 : 1000, Ak098372, Abcam, UK), Abcb1a (1 : 1000, ab3373, Abcam, UK), IL-1β (1 : 1000, S328, NIBSC, UK), IL-6 (1 : 1000, Tyr705, Abcam, UK), IL-8 (1 : 1000, S333, Abcam, UK), TNF-α (1 : 1000, C432, PA, USA), Bcl-2 (1 : 1000, ab32124, Abcam, UK), Bax (1 : 1000, 6A7, Abcam, UK), caspase-3 (1 : 1000, H-277, Abcam, UK), and β-actin (1 : 1000, AC15, Abcam, UK). HRP-labeled goat anti-rabbit IgG antibody (Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China, 1 : 5000) was then incubated with cells. The blots were developed with the ECL and quantified with Quantity One v4.6.2 software.