Protein g coupled sepharose beads

Wild-type, SKAP55^−/−, or ADAP^−/− CD8⁺ CTLs were left unstimulated or stimulated with 10 nM OVA_257-264-pulsed EL-4 for 4 h. Cell pellets were lysed for immunoblotting with anti-mouse NFATc1 and β-actin. For immunoprecipitation, CD8⁺ CTLs were stimulated with hydrogen peroxide and sodium orthovanadate. Cell lysates were incubated with various antibodies and protein G-coupled sepharose beads (Sigma) respectively, followed by immunoblotting with antibodies against SKAP55, ADAP, and Fyn.

Cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) in the presence of 1% protease inhibitor cocktail and 1% phosphatase inhibitors (Sigma‐Aldrich). Samples were separated using SDS‐PAGE, transferred onto polyvinyl difluoride (PVDF) membrane, and blocked with 5% BSA at room temperature for 2 h. Membranes were then incubated with indicated primary antibodies, followed by incubation with horseradish peroxidase (HRP)‐conjugated secondary antibodies (Proteintech). For immunoprecipitation, Cell lysates were incubated with various antibodies and protein G‐coupled sepharose beads (Sigma) or Urolithin A magnetite microspheres, respectively, followed by immunoblotting with indicated antibodies. All antibodies for western blot are listed in Table S2 (Supporting Information).