Anti shp2

Spleen proteins were extracted using RIPA lysis buffer (Solarbio, Beijing, China) and centrifuged at 12,000 g for 10 min at 4°C. Protein concentration was assessed using the BCA Protein Assay Kit (Biosharp, Beijing, China). Samples containing equal amounts of protein (80 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to 0.45 μm polyvinylidene difluoride membranes (Biosharp, Beijing, China). The non-specific binding sites on the membrane were then blocked with 5% fresh non-fat dry milk in Tris buffered saline TBS with Tween (10 mM Tris, 150 mM NaCl, pH7.4) with 0.1% Tween 20) for 2 h. Subsequently, they were incubated with primary antibodies (anti-IL-2, anti-STAT1, anti-SHP2, anti-JAK1, and anti-p-JAK1; 1:1000 dilution; Abcam, Cambridge, UK) and primary β-actin antibody (1:1,000 dilution; abmart, Shanghai, China) overnight at 4°C, respectively. After this, the membrane was washed followed by three 10 min washes in TBST, and then incubated with anti−mouse secondary antibody (1:2000 dilution; Cell Signaling Technology, Shanghai, China) for 2 h at room temperature followed by three 10 min washes in TTBS. Finally, the chemiluminescent signals were observed with a multifunctional gel imaging system (Bio-Rad, USA) and densitometry for immunoreactive bands was performed with National Institutes of Health software (Image J).

To investigate the interaction between 2B4 and SHP-2 or Fyn in the infected group with or without anti-2B4 antibody treatment, we used an anti-2B4 (Cell Signaling Technology, USA), anti-SHP-2 (Abcam, England), or anti-Fyn antibody (Abcam, England) for immunoprecipitation (IP). Cells from the uninfected group, infected group, and anti-2B4 antibody-treated infected group were collected and lysed in IP lysis buffer (Beyotime Technology, China) supplemented with protease inhibitors (Beyotime Technology, China) on ice for 45 min and centrifuged at 12,000 rpm at 4 °C for 15 min. Next, the supernatants were collected and incubated with 1 µg of the appropriate antibody at 4 °C overnight and precipitated with protein A/G-agarose beads (Beyotime Technology, China) for another 7 h at 4 °C. The beads were then washed with lysis buffer three times by centrifugation at 12,000 rpm at 4 °C. The precipitated proteins were denatured in 5 × loading buffer and analyzed by Western blotting.

Mouse rIL‐21 and granulocyte–macrophage colony‐stimulating factor (rGM‐CSF) were purchased from Peprotech (Rocky Hill, NJ, USA). All flow cytometry antibodies and Cytofix/Cytoperm Kits were purchased from BD Pharmingen (San Diego, CA, USA). The anti‐SHP‐2, PTPN‐22, DUSP5/6, and β‐actin antibodies used in Western blotting were purchased from Abcam (Cambridge, MA, USA). Quantikines were purchased from R&D System (Minneapolis, MN, USA). The CD3‐CD28 beads, cell trace, and Trizol were purchased from Invitrogen (Burlington, ON, Canada). The cell Lytic M buffer and lipopolysaccharide (LPS) were purchased from Sigma (St‐Louis, MO, USA). T‐cell enrichment kits were purchased from StemCell Technologies (Vancouver, BC, Canada). Peptides were synthesized by GenScript (Piscataway, NJ, USA).