Extended Data Fig. 7: Purification and analysis of SHMT2 mutants.

a, Elution profile of the indicated forms of SHMT2ΔN from an S75 10/300 size-exclusion chromatography column (single experiment). b, Coomassie-stained SDS–PAGE analysis of the indicated SHMT2ΔN protein preparations (data are representative of two independent experiments). c, BRISC DUB activity against a fluorogenic K63-linked diUb substrate in the presence of the indicated SHMT2ΔN mutants. Data are mean ± s.e.m. of three independent experiments carried out in duplicate. d, BRISC DUB activity against K63-linked hexaUb chains in the presence of the different forms of SHMT2ΔN, or SHMT1. e, Ubiquitylation levels of IFNAR1 after IFNα stimulation in HEK293T cells that overexpress the indicated forms of Abraxas 2 and SHMT2ΔN. The annotation ‘LL->RR’ denotes SHMT2ΔN(L211R/L215R). IFNAR1 immunoprecipitation (IP) was performed under denaturing conditions and ubiquitin levels were detected using the vu-1 antibody. Mock IP was performed using a generic rabbit IgG antibody. f, Immunoprecipitation (IP) performed using anti-Flag antibody in MEFs that were transiently transfected with Flag–HA epitope-tagged SHMT2ΔN or mutants. Immunoblot was performed for Abraxas 2 and SHMT2, as indicated. UTF, untransfected cells (used as control). g, MEFs that overexpress the indicated SHMT2ΔN or mutants were challenged with LPS, and interferon-receptor-dependent signal transduction response was assessed by immunoblot for STAT1 phosphorylated at Y701. Data shown in d, e are representative of three independent experiments. For gel source data, see Supplementary Fig. 1.