Hrp conjugated anti rabbit secondary antibody

Western blot was used to detect the expression level of HER2 in 4T1-Luc-HER2 cells and the expression level of CAR in the CAR-transduced T cells. Cells were lysed in lysis buffer, and the protein concentrations of cell lysates were analyzed by the BCA kit (Beyotime Institute of Biotechnology, Shanghai, China). Lysates (10 μg per lane) were resolved by 10% SDS-PAGE, transferred to PVDF membranes (EMD Millipore). The blots were blocked with 5% non-fat dry milk in TBST, immunoblotted with anti-human HER2 antibody (dilution 1:1,000; Abcam) or anti CD3ζ polyclonal antibody (dilution 1:1,000; Affinity Biosciences, Jiangsu, China) and anti-rabbit HRP-conjugated secondary antibody (dilution 1:5,000; Santa Cruz Biotechnology, Inc. CA, USA), developed utilizing ECL reagent (Beyotime Institute of Biotechnology), and detected using the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.). The expression level of HER2 or CAR (containing exogenous CD3ζ) was detected, respectively, using tubulin or endogenous CD3ζ as a loading control.

Whole cell extracts were obtained by incubating cells in modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% NP-40, 0.25% sodium-deoxycholate, 150 mM NaCl, and 1 mM EDTA) supplemented with a protease inhibitor cocktail (Complete, Roche) and phosphatase inhibitors (2 mM Na₃VO₄ and 2 mM NaF) for 30 min on ice, then cleared by centrifugation at 13,000 × g for 10 min at 4 °C. Total protein content (10–30 μg) was separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (Hybond, GE Healthcare) for Western blotting. Primary antibodies were incubated overnight at 4 °C in PBS + 5% BSA and were: rabbit anti-p44/42 MAPK (Erk1/2, Cell Signaling Technology) used at 1:1000, rabbit anti-Phospho-p44/42 MAPK (P-Erk1/2, Thr202/Tyr204, Cell Signaling Technology) used at 1:1000. Anti–rabbit HRP-conjugated secondary antibody (Santa Cruz Biotechnology) was used at 1:10,000 in PBS + 5% BSA for 1 h at room temperature, and HRP activity was visualized using the ECL Plus system (BioRad). Images were quantified using the “Analyze Gel” function of ImageJ.

Cells were lysed in RIPA buffer, and protein concentration was determined with the Bio-Rad Protein assay using protein assay dye reagent concentrate (Bio-Rad, cat. #500-0006). Total lysates were separated on a 10 % SDS-PAGE gel for 1 h at 200 V. Proteins were subsequently transferred to a nitrocellulose membrane via semi-transfer for 25 min at 20 V, blocked with 5 % milk in TBS-Tween for 1 h at room temperature, and then incubated with the appropriate primary antibody at 4 °C overnight. Primary antibodies used were: rabbit polyclonal to vimentin (Abcam, cat. #ab45939), rabbit polyclonal to keratin 14 (Covance, cat. #PRB-155P), and rabbit polyclonal to actin (Abcam, cat. #ab8226). Membranes were then incubated in blocking buffer plus anti-rabbit HRP-conjugated secondary antibody (Santa Cruz, cat. #SC2054) for 1 h at room temperature and exposed using the LumiSensor™ chemiluminescent HRP substrate kit (GenScript, cat. #L00221V500).

ELISA plates were coated with clarified cell culture supernatant containing VLPs. After blocking (3% BSA in phosphate-saline buffer [PBS] 0.05% Tween 20 buffer) and washing, rabbit monoclonal anti-ZIKV (strain SPH2015) envelope protein (Domain III) antibody (1:2,000 dilution) was added for 2 h at RT. Next, after washing an anti-rabbit HRP-conjugated secondary antibody (Santa Cruz Biotechnology) (1:1,500) was added. TMB Substrate Solution (Thermo Fisher Scientific) was used for reaction visualization. The reaction was stopped with 0.5 M H₂SO₄, and the absorbance was measured at 450 nm with a microplate reader (TECAN).

A total of 30 μg of protein lysates was separated by 12% SDS-PAGE and then transferred to PVDF membrane (Pall, Germany) by semidry electroblotting at constant voltage (25 V) for 60 min. The membranes were blocked with 5% BSA in 1x TBS-N for 1 hr and then incubated with anti-S100A2 primary antibody (Abcam, UK) at 4°C overnight. The blots were washed three times for 5 min with TBS-N buffer and incubated with anti-rabbit HRP-conjugated secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at room temperature for 30 min. Signals were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, USA).