Themis acts to increase phosphorylation of Shp1.

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Themis acts to increase phosphorylation of Shp1. Themis acts to increase phosphorylation of Shp1. (A) Western blot with immunoprecipitated Shp1 assessing levels of p-Shp1 associated with Shp1 from Themis+/+ and Themis−/− thymocytes under both unstimulated and stimulated conditions using biotinylated antibody cross-linking with streptavidin. (B) Tyrosine phosphatase activity assay of immunoprecipitated Shp1 from OT-I β2m−/−Themis−/− and OT-I β2m−/−Themis+/+ thymocytes with or without additional recombinant His-tagged Themis in untreated and treated samples with AP for 1 h at 37 °C before doing the assay. **P < 0.005; ***P < 0.001. (C) Western blot with immunoprecipitated Shp1, assessing levels of p-Shp1 associated with Shp1 in similar samples as used for the phosphatase assay shown in B. IB, immunoblot. Raw values of p-Shp1 and Shp1 were calculated using LI-COR Odyssey software with the help of the background subtraction method. Normalized values were calculated by taking the ratio of raw values of p-Shp1 and Shp1 from the same samples. Monika Mehta et al. PNAS 2018;115:48:E11331-E11340 ©2018 by National Academy of Sciences