Skimmed milk

ELISA was used to analyze ORF132 specific antibody induced by EBY100/pYD1-ORF132 in crucian carp serum. The ELISA plate was coated with 1 µg/mL fish serum and blocked with PBS containing 5% skimmed milk (Sangon, Shanghai, China) for 2 h at 37 °C. After that, the plates were incubated with 1 µg ORF132 amino acid residue (KKKKKTTKKSSSKSSGKNKRSGGYDN) for 1 h at 37 °C. ORF132 polyclonal antibody from mouse was custom made in GL Biochem (Shanghai, China) using amino acid residue (KKKKKTTKKSSSKSSGKNKRSGGYDN). Next, 100 µL ORF132 polyclonal antibody (1:2000 diluted) was added to each well and incubated 1 h at 37 °C. Next, HRP-conjugated Goat Anti-mouse IgG (1:5000 diluted) (Beyotime) was used to recognize ORF132 polyclonal antibody. Finally, TMB (TianGen) was used as colorimetric substrate and the plates were read at 450 nm with a microplate reader (Potenov, Beijing, China).
ORF132 was analyzed by ABCpred and DNAStar, antigenic epitope fragment rich region (amino acids 127–152, KKKKKTTKKSSSKSSGKNKRSGGYDN) was selected to produce ORF132 polyclonal antibody. ORF132 polyclonal antibody was custom made in GL Biochem Ltd. (Shanghai, China) by peptides amino acids 127–152.

Rabbit anti-human antibody CHSY3 (100 μg) was obtained from OriGene China. The primary antibody to β-actin and CD3+/CD4+/CD8+ T cells alpha rabbit monoclonal antibody were provided by Cell Signaling Technology, Inc. (Danvers, MA, United States). Horseradish peroxidase (HRP)-conjugated anti-rabbit antibody was provided by Jackson ImmunoResearch Inc. (West Grove, PA, United States). Bovine serum albumin was obtained from Sigma-Aldrich (St. Louis, MO, United States). Skimmed milk and Tween-20 were purchased from Sangon Biotech Co., Ltd. (Shanghai, China).

The antigens were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane as described previously [24 (link)]. The membranes were washed three times for 5 min each in TBST (20 mM Tris–HCl, 150 mM NaCl, 0.05% [V/V] Tween 20, pH 7.4) at RT, coated in 5% skimmed milk (Sangon Biotech) for 2 h and incubated overnight with sera from mite-allergic mice (diluted 1:5 with 0.01 M PBS). The membranes were washed four times and incubated with biotinylated anti-mouse IgE antibody (diluted 1:600 with PBS; BioLegend) for 1 h at 37 °C. Next, the membrane was washed and incubated with HRP–streptavidin (diluted 1:800 with PBS; BioLegend) for 30 min at 37 °C. Lastly, the membrane was washed four times, and protein signals were detected using diaminobenzidine (TIANGEN).

The rabbit anti-human CDK16 antibody was purchased from CUSABIO (CSB-PA017648ESR2HU) (100 μl, Wuhan, China). A horseradish peroxidase (HRP)-conjugated anti-rabbit antibody was purchased from Jackson ImmunoResearch Inc. (West Grove, PA, United States). Bovine serum albumin was purchased from Sigma-Aldrich (St. Louis, MO, United States). Skimmed milk and Tween-20 were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The TRIzol reagent was purchased from Thermo Fisher Scientific (United States). The PrimeScriptTM First Strand cDNA Synthesis Kit was purchased from TaKaRa (Tokyo, Japan), and the SYBR Green Real-Time PCR Master Mix was purchased from TOYOBO (Osaka, Japan).