TOX inhibits fatal autoimmune hepatitis by mitigating IL-17A production of γδ T cells via stabilization of TCF-1
Description
Proteins from DN2 thymocyte lysis derived from 2 week-old wild-type mice were immunoprecipitated with anti-TOX antibody, separated by SDS-PAGE, and then visualized with Coomassie brilliant blue staining. Protein in-gel digestion and LC-MS/MS analysis were performed and the resulting spectra were mapped to Mus_musculus_Uniprot database by the search engines: Proteome Discoverer 2.2 (PD 2.2, Thermo). Our data showed that TOX combined with TCF1, thereby exerting the function of stabilization for TCF1 and regulating the development of thymocytes and inflammation in mice.
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Proteins from DN2 thymocyte lysis derived from 2 week-old wild-type mice were immunoprecipitated with anti-TOX antibody, separated by SDS-PAGE, and then visualized with Coomassie brilliant blue staining. Protein in-gel digestion and LC-MS/MS were performed as follows: 100 μL with 100 mM TEAB, 1ug trypsin and 1/300 CaCl2 were added into gel, proteins were digested overnight at 37°C. After centrifugation in low speed, the supernatant was collected, 200 μL of acetonitrile was added, put under vortex and mixed. And the supernatant was extracted in 100 μL of 0.1% formic acid (FA). Combined the supernatant and centrifuged at 12000 g for 5 min at room temperature and then lyophilized. The powder was dissolved and mixed in 0.1% of formic acid. The supernatant was slowly loaded to the C18 desalting column, washed with washing solution (0.1% formic acid, 3% acetonitrile) 3 times, then eluted twice by some elution buffer (0.1% formic acid, 70% acetonitrile). The eluents were collected and lyophilized. Peptides were separated in a home-made analytical column (15cm×150 μm, 1.9 μm), using a linear gradient elution as listed in Table 1. The separated peptides were analyzed by Q ExactiveTM series mass spectrometer (Thermo Fisher), with ion source of Nanospray Flex™（ESI）, spray voltage of 2.1 kV and ion transport capillary temperature of 320°C. Full scan range from m/z 350 to 1500 with resolution of 60000 (at m/z 200), an automatic gain control (AGC) target value was 3×106 and a maximum ion injection time was 20 ms. The top 40 precursors of the highest abundant in the full scan were selected and fragmented by higher energy collisional dissociation (HCD) and analyzed in MS/MS. The resulting spectra were searched against Mus_musculus_Uniprot database by the search engines: Proteome Discoverer 2.2 (PD 2.2, Thermo).