Horseradish peroxidase conjugated anti mouse antibody

The separated proteins in a 10% SDS-polyacrylamide gel were blotted on a polyvinylidene fluoride (PVDF) membrane. The primary and secondary antibodies were monoclonal anti-CD36 antibody (Santa Cruz, cat. sc-7309) and 1:10,000 diluted horseradish peroxidase conjugated anti-mouse antibody (Thermo Scientific, cat. 31430), respectively.

Recombinant MBP-NbWRKY40, MBP-NbWRKY8, or MBP alone from E. coli lysates were immobilized on amylose resins (NEB), incubated for 1 h at 4°C with purified GST-XopS or GST alone, eluted, and analyzed by immunoblotting using either anti-GST antibody (1:1,000, horseradish peroxidase-conjugated; Santa Cruz Biotechnology Inc.; cat. no. sc-138 HRP) or anti-MBP antibody (1:10,000; NEB, cat. no. E8032S; used with 1:10,000 secondary horseradish peroxidase-conjugated anti-mouse antibody; ThermoFisher; cat. no. 31430).

Synchronized L4 larvae N2 worms washed with PBS to clear residual bacteria were grown on plates with OP50 or S. Paratyphi A for 12 h. Worms (KU25 pmk-1(km25) IV was analyzed to verify mutant) were rinsed by PBS and then lysed in the RIPA buffer containing protease and phosphatase inhibitor mixture prior to triturating. BCA Protein Assay Kit (Beyotime) was applied to quantify the concentration of protein. After boiled, samples containing 20 μg proteins were separated on 12% SDS–PAGE and electro-transferred onto 0.2 μm polyvinylidene difluoride membranes. After blocked in 5% fat-free milk, The membranes were incubated with primary antibodies against phospho-p38/PMK-1 (1:1000, Cell Signaling) or actin (1:5000, Cell Signaling) followed by horseradish peroxidase-conjugated anti-mouse antibody (1:5000, Thermo Fisher Scientific). Quantification of blot were analyzed using Image J.