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. 2023 Apr 17;220(7):e20221649. doi: 10.1084/jem.20221649

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Figure 2. — TCR signaling responses are impaired and SHP1 tyrosine phosphatase activity is increased in Grb2-deficient thymocytes. (A and B) CD3+CD4 antibody activation of DP enriched +/+, Themis^−/−, and CD2-iCre;Grb2^f/f thymocytes. Thymocytes were rested for 6 h at 37°C, pre-incubated with biotinylated anti-CD3 and anti-CD4 antibodies, then streptavidin was added for the indicated times at 37°C. Cells were lysed at 4°C and proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes then blotted with the indicated antibodies. Results shown are representative of three experiments. (C) Oxidized SHP1 in Themis^−/− and CD2-iCre;Grb2^f/f thymocytes following treatment with H₂O₂. SHP1 (arrowhead) was identified by its mobility on SDS-PAGE. (D) Summary of experiments as shown in C. +/+ was arbitrarily set to 100% for comparison. Error bars show SD. ***P < 0.005. T test, two tailed, Type 2. Data shown are from four experiments. (E and F) GRB2 and THEMIS cooperate to inhibit SHP1 tyrosine phosphatase activity. HEK-293 cells were transfected with the indicated plasmids and Western blots of cell lysates were performed with the indicated antibodies. Arrowhead designates endogenous GRB2. Source data are available for this figure: SourceData F2.