PTPN22 KO Jurkat cells overexpressing 3× FLAG-tagged WT or S751A mutant were treated with 20 μM CHX for the indicated times. Cells were harvested and washed twice in PBS buffer with 1% FBS. After staining with Viability Dye eFluor 780 for 30 min on ice, cells were fixed with intracellular fixation buffer (Invitrogen) for 15 min at room temperature (RT). After incubating with Fc block (BD Biosciences) for 15 min at RT, cells were stained with monoclonal anti-FLAG M2-FITC Ab for 1 hour at RT. After washing three times with fluorescence-activated cell sorting (FACS) buffer, cells were analyzed using a ZE5 flow cytometer (Bio-Rad).

Jurkat PTPN22 S751A KI cells were starved for 24 hours and added into 24-well plates, which were precoated with CD3 Ab (5 μg/ml). Cells were stimulated for 4 hours at 37°C together with CD28 Ab (5 μg/ml) in RPMI 1640 basic medium. Cells were harvested and washed twice in PBS buffer with 1% FBS. Next, cells were incubated with Fc block for 15 min at RT. After staining with APC-conjugated anti-human CD69 Ab and Viability Dye eFluor 780 for 30 min on ice, cells were washed three times with FACS buffer and analyzed using a flow cytometer. Results were analyzed using FlowJo software (Treestar, Ashland, OR).

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