Extended Data Fig. 3: SHP1 dephosphorylates THEMIS at Tyr34 site.
From: THEMIS is a substrate and allosteric activator of SHP1, playing dual roles during T cell development
a, In vitro DiFMUP assay, which detects the activity of protein phosphatase SHP1 showed that the constitutively activated SHP1-EA (E74A) mutant has the highest activity; the lowest activity was detected for the phosphatase-dead SHP1-CS (C453S) mutant (n = 5 biologically independent samples). b, Dot blot results indicate that the constitutively activated SHP1-EA mutant can dephosphorylate an artificial THEMIS p-Y34 peptide; the peptide sequence is shown above. c, In vitro dephosphorylation assays which mixed the purified SHP1 protein from E. coli and the THEMIS protein affinity purified from HEK293T cells at 30 °C for 1 hour. Western blotting analysis indicated that SHP1 catalyzed the dephosphorylation of THEMIS Tyr34. d, Src kinase family inhibitor PP2 treatment contributed to the blockade of phosphorylation transduction, including THEMIS Tyr34 site after TCR signaling activation by C305 (anti-TCR) antibody.
