Mouse anti ha antibody

For cultured cell samples, the culture supernatant, washing buffer and cell suspension were all harvested. The pelleted cells were lysed in RIPA buffer and centrifuged. The supernatants were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was first incubated with anti-human HA antibody (1:100; cat# 3724, Cell Signaling Technology), or cleaved caspase-9 antibody (1:1000; cat# 9508, Cell Signaling Technology), or cleaved caspase-3 antibody (1:1000; cat# 9661, Cell Signaling Technology), or β-actin antibody (1:2000; cat# TA811000, OriGene) overnight at 4 °C, and subsequently with HRP-conjugated appropriate secondary antibody. The protein bands were developed with ECL reagent, detected by the ChemiDoc MP imaging system (Bio-Rad), and were analyzed using the Image Lab 5.0 software (Bio-Rad). In animal experiments, after mice were euthanized, SN and striatum tissues from both sides of the brain were dissected and lysed in RIPA lysis buffer. The remaining steps were the same as that for cultured cell samples, except for the primary antibody being mouse anti-HA antibody (Beyotime) or anti-GAPDH antibody (Proteintech).

To examine the secretion of candidate effector proteins, overnight cultures of wild-type E. piscicida strain EIB202, the T3SS mutant, and the T6SS mutant expressing HA-tagged effectors were subcultured at 1:100 in DMEM and grown without shaking for 12 h at 30°C. L-Arabinose was added to induce the expression of effector-HA when the optical density (OD)₆₀₀ was 0.6. The supernatant and the total bacterial proteins were prepared as described previously (Hou et al., 2017 (link)). The supernatant and the total bacterial pellet fraction were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane for immunoblotting. Membranes were probed with mouse anti-HA antibody (1:2,000, Beyotime, China) and rabbit anti-RNAP antibody at 1:2,000 followed by horseradish per-oxidase (HRP)-conjugated goat anti-rabbit IgG at 1:5,000 (Beyotime, China).