Camkkβ

GYY4137 [morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate], DTT (DL-dithiothreitol), AMPK inhibitors Compound C (6-[4-[2-(1-piperidinyl)eth-oxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine), Ara-A (ATP-mimetic, 9-β-D-arabinofuranoside), and MMTS (S-methyl methane thiosulfonate) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Biotin-HPDP and HRP (horseradish peroxidase)-conjugated streptavidin were purchased from Thermo Scientific (Rockford, IL, United States). Primary antibodies for phospho-AMPKα (Thr172), AMPKα, phospho-CaMKKβ (Ser458/495), and CaMKKβ were bought from Cell Signaling Technology (Boston, MA, United States). Primary antibodies for NeuN (neuron-specific nuclear protein), c-Fos, and NPY were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Primary antibody for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HRP-conjugated secondary antibodies, fluorescent secondary antibodies, and bovine serum albumin (BSA) were obtained from Beyotime Biotechnology (Shanghai, China). Other agents were all purchased from commercial suppliers. Biotin-HPDP was freshly dissolved in dimethysulfoxide (DMSO) and other drugs were prepared freshly with double-distilled water or buffer solutions to the final concentrations before application.

RAW 264.7 cells were lysed with RIPA reagents (Pierce, Rockford, IL, USA) containing cocktail protease and phosphatase inhibitors (Roche, Switzerland). Plasma proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Blots were blocked with 5% BSA for 1 h and incubated with primary antibodies (1 : 1000 dilutions) for CaMKKβ, pCaMKKβ, AMPKα, pAMPKα, SIRT1, IκBα, IRF3, pIRF3, p38, p-p38, and tubulin (Cell Signaling, USA) at 4°C overnight. Then, the blots were further incubated with HRP-conjugated secondary IgG antibodies (1 : 2000 dilutions, Cell Signaling) at 37°C for 1 h. Chemiluminescence images were developed with SuperSignal Sensitivity Substrate Kit (Pierce) with a ChemiDoc XRS imaging system (Bio-Rad, USA).