Anti cd3ζ antibody

Western blot was performed to verify the expression of CAR proteins. The samples were immuno-blotted with a goat anti-human CD3ζ antibody (Santa Cruz Biotechnology). The blot was then incubated with HRP-conjugated rabbit anti-goat IgG (Sigma). The expression of Bcl-xL in CAR T cells after they were co-cultured with tumor cells for 24 h at an effector: target ratio of 3:1 was examined by western blot using an anti-Bcl-XL antibody (Cell Signaling Technology, USA). β-Actin was used as a loading control.
The expression of human CD3ζ and GPC3 proteins in tumor tissues was examined by western blot using an anti-CD3ζ antibody (Santa Cruz Biotechnology) and an anti-GPC3 antibody (mAb1G12, BioMosaics Inc). Solutions of diluted antibody in PBS-T buffer were supplemented with 5% nonfat dry milk (primary antibody diluted 1:1000; secondary antibody diluted 1:2000). The blots were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Kangchen Biotech, Shanghai, China), and the proteins were detected using an ECL western blot analysis system (Pierce, Thermo Scientific, Rockford, IL) in accordance with the manufacturer's instructions.

Equal masses of protein lysates were loaded onto a 4–20% SDS-PAGE gel for electrophoresis. HM2 was used to detect GPC1 expression at 1 μg/ml. An anti-CD3ζ antibody was purchased from Santa Cruz Biotechnology (Cat#sc-166435, 1:200 dilution). Antibodies including anti-ZAP70-pTyr319 (Cat#2701S, 1:500 dilution), anti-LAT-pTyr220 (Cat#3584S, 1:500 dilution), anti-SLP-76-pSer376 (Cat#14745S, 1:500 dilution), anti-PLC-γ1-pTyr783 (Cat#2821S, 1:500 dilution), anti-RelA/p65-pSer536 (Cat#3033S, 1:500 dilution), anti-RelB (Cat#10544S, 1:500 dilution), anti-β-catenin (Cat#9582S, 1:1000 dilution), anti-β-actin (Cat#8457S, 1:2000 dilution), GAPDH (Cat#5174S, 1:5000 dilution), and anti-PCNA (Cat#2586S, 1:1000 dilution) were obtained from Cell Signaling Technology. Anti-Wnt3a was purchased from Abcam (Cat#ab28472, 1:100 dilution). Band intensities were quantified using ImageJ (NIH) and normalized to a mock sample.

Jurkat T cells were cultured in RPMI supplemented with 10% FBS, 50 μM β-mercaptoethanol and 1% penicillin-streptomycin in the presence or absence of 0.5 mM sucralose for 48 h. Cells were washed once with PBS and resuspended at the concentration of 30 × 10⁶ cells ml^–1 in DPBS containing 1% FBS, 5 mM glucose, 2 mM glutamine, 50 μM β-mercaptoethanol and 1% penicillin-streptomycin in the presence or absence of 0.5 mM sucralose.
Cells were kept on ice and activated with 5 μg ml⁻¹ anti-CD3 (OKT3) at 37 °C as indicated. The reaction was stopped by adding ice-cold PBS. Proteins were extracted using immunoprecipitation lysis buffer containing 0.2% Triton X-100, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, protease inhibitor cocktail (Thermo Scientific) and phosphatase inhibitor cocktail (Cell Signaling) at 4 °C for 30 min. Total protein content was quantified using a BCA assay. CD3ζ was immunoprecipitated from 1.5 mg total protein using an anti-CD3ζ antibody (Santa Cruz), using 4 μg antibody per 0.5 mg total protein and Protein A/G plus Agarose (Thermo Fisher) for 2 h at 4 °C. Immunoprecipitated proteins were washed three times with immunoprecipitation lysis buffer and western blots were performed as indicated.