Hy k0011

Proteins of primary microglia or primary astrocyte were isolated using RIPA lysis buffer containing protease (HY-K0011, MedChemExpress, NJ, USA) and phosphatase inhibitor cocktail (HY-K0022, MedChemExpress, NJ, USA). After being uniformed and denatured by boiling at 95 °C for 5 min, protein samples were loaded, processed for sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Then the membranes were blocked with 10% skimmed milk at room temperature for 1 h followed by incubating with primary antibodies against β-actin (66009-1-Ig, Proteintech, Chicago, IL, USA), PKM2, PKM1 (15821-1-AP, Proteintech, Chicago, IL, USA), C3, p-p65 ser536, p-p65 ser468 (3039, Cell Signaling Technology, Danvers, MA, USA) and p65 (6956, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. After being washed with TBST, the membranes were incubated with corresponding anti-mouse (SA00001-1, Proteintech, Chicago, IL, USA) or anti-rabbit (SA00001-2, Proteintech, Chicago, IL, USA) second antibody at room temperature for 1 h and visualized by ECL chemiluminescent substrate reagent.

A. tumefaciens strain GV3101 carrying the pFGC5941-MYC-ABI5 and pFGC1008-MYB44-FLAG plasmids were infiltrated into the N. benthamiana leaves. After inoculation for 2 d, total proteins were isolated with isolation buffer [50 mM Tris–HCl (pH 8.0), 1 mM EDTA, 150 mM NaCl, 0.2% Triton X-100 (Solarbio, T8200), 1 mM phenylmethylsulphonyl fluoride (Solarbio, P8340), 1 protease inhibitor cocktail tablet (MedChemExpress, HY-K0011)], and were centrifuged at 12000 r at 4°C for 15 min. The proteins were immunoprecipitated with anti-FLAG magnetic beads (MedChemExpress, HY-K0207) according to the manufacture’s instruction. For analysis the stability of MYB44, the N. benthamiana leaves were injected with 0.1 mM MG132 for 2 h, and then the leaves were harvested.