Anti t akt

Piperine (PIP) specified to be more than 97% pure was purchased from Shanghai Winherb Medical Science Co. (Shanghai, China).¹³ Lactate dehydrogenase (LDH) and creatine kinase (CK) were detected by commercially available ELISA kits (Jiancheng Bioengineering Institute). The following primary antibodies were from Abcam: anti‐p‐PI3K (1:600 dilution, ab182651), anti‐t‐PI3K (1:400, ab191606), anti‐NLRP3 (1:1000, ab263899), anti‐IL‐18 (1:500, ab191860) and anti‐GAPDH (1:1000, ab37168). The following primary antibodies were obtained from Cell Signaling Technology: anti‐p‐AKT (1:800, #9145), anti‐t‐AKT (1:600, #9139), anti‐cleaved caspase‐1(1:600, #89332), anti‐pro‐caspase‐1 (1:1000, #24232), anti‐cleaved IL‐1β (1:500, #63124) and anti‐pro‐IL‐1β (1:500, #12703). Antibody against RP105 (1:600, PAB18126) was obtained from ABNOVA. The BCA protein assay kit was purchased from Pierce. Evans blue and TTC dying were purchased from Beyotime Institute of Biotechnology. LY294002 (a PI3K/AKT inhibitor) were from Sigma‐Aldrich. The miR‐383 mimic and its scrambled oligonucleotides (miR‐NC) were produced by GenePharma Co., Ltd.

Approximately 40 μg of protein was separated on a 10% SDS gel, following which it was transferred to a nitrocellulose membrane. The nonspecific binding sites of the nitrocellulose membrane were blocked using 5% nonfat milk and then incubated with anti-BMAL1 (Abcam) (1:1000), anti-T-AKT (Cell Signaling Technology, Danvers, MA, USA) (1:1000), anti-P-AKT (Ser473) (Cell Signaling Technology) (1:1000), and anti-GAPDH (Abcam) (1:5000) antibodies at 4 °C overnight. After washing, the blot was incubated and diluted with the corresponding peroxidase-conjugated secondary antibodies for 1 h at room temperature. Finally, the protein signals were detected using the enhanced chemiluminescent detection system (Millipore, Billerica, MA), and the ratio of a target protein to that of the intensity of GAPDH was obtained as each target protein level.

Proteins of hippocampus tissues were extracted using RIPA buffer (1% Nonidet P-40, 0.1% SDS, 150 mM NaCl, 50 mM Tris–HCl pH 7.5, and 0.5% deoxycholate) with 10% of phosphatase inhibitor and protease inhibitor (Roche, Basel, Switzerland). Proteins were loaded on SDS-PAGE gel and run by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membrane. Membrane blocked by 5% skim milk for 1 h, and incubated with primary antibody including anti-pERK (9102, Cell signaling Technology, Beverly, MA, USA), anti-tERK (9101s, Cell signaling Technology), anti-BDNF (ab108319, Cambridge, MA, USA), anti-pCREB (9198, Cell signaling Technology), anti-tCREB (9197, Cell signaling Technology), anti-pAKT(9271, Cell signaling Technology), anti-tAKT (9272s, Cell signaling Technology), and anti-Actin (sc-1616, Santa Cruz, CA, USA) antibodies at 4 °C for overnight. Anti-IgG horseradish peroxidase antibody (Pierce Biotechnology, MA, USA) correspond with the host of primary antibody was used as secondary antibody. Proteins band was detected by ECL system (Thermo Scientific, Inc.). The quantification of band intensity was normalized with actin using Image J program (version 1.52c; National Institutes of Health, Bethesda, MD, USA).

Cells were lysed by boiling in SDS-PAGE sample loading buffer (1% SDS, 10% glycerol, 10 mM Tris-Cl, pH 6.8, 1 mM EDTA). As positive control for Akt phosphorylation, cardiomyocytes were treated with 100 nM insulin. The samples were resolved by SDS-PAGE in 10% acrylamide gel. Phosphorylated and non-phosphorylated Akt was compared to the content of the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH). To evaluate phosphorylation status of MAPK/ ERK1/2, phosphorylated and non-phosphorylated ERK1/2 was compared to the content of the housekeeping protein alpha-tubulin.
Primary antibodies used were anti-tAkt (Cell Signaling Technology, Inc. Cat. no. 2920) anti-pAkt (Phospho-Serine 473, Cell Signalling Technology, Inc. Cat. no. 4060), anti-tERK1/2 (Cell Signalling Technology, Inc. Cat. no. 9107), anti- pERK1/2 (Phospho-Threonine 202/Phospho-Tyrosine 204, Cell Signalling Technology, Inc. Cat. no. 4370), anti-GAPDH (abcam, Cat no. ab9485) and anti-alpha tubulin (abcam, Cat no. ab4074) All primary antibodies were used at a dilution of 1:1000. Following overnight incubation, the membrane was washed and probed with secondary antibodies using standard Western blot protocol.

GH3 cells in 10-cm plates were lysed in radio-immunoprecipitation assay buffer (20 mM Tris, 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 5% phosphatase inhibitors, 0.5% protease inhibitors), and the protein content was measured using a Coomassie (Bradford, UK) Protein Assay Kit (Pierce, Rockford, IL, USA). Equal amounts of protein (10 mg) were heat-denatured in 2 × sample buffer (2% SDS, 62.5 mM Tris, pH 6.8, 0.01% bromophenol blue, 1.43 mM mercaptoethanol, and 0.1% glycerol), separated on a 10% SDS–polyacrylamide gel, transferred onto polyvinylidene difluoride membranes (Bio-Rad), and blotted with the appropriate antibodies: anti-GH (Santa Cruz Biotechnology, Dallas, TX, USA), anti-tAKT (Cell Signaling Technology, Danvers, MA, USA), anti-pAKT (Cell Signaling Technology), anti-mTOR (Cell Signaling Technology), anti-pCREB (Cell Signaling Technology), anti-tCREB (Cell Signaling Technology), and anti-β-actin (Santa Cruz Biotechnology). Immunocomplexes were detected using an enhanced chemiluminescence system (Cell Signaling Technology)^{43 (link)}.