4 hydroxy 3 nitrophenylacetyl

Insulin-A-chain derived peptides (InsA) (Peptides&Elephants, Berlin) were dissolved according to their water solubility. For covalent coupling of peptides to key hole limpet hemocyanin (KLH) the N-terminal cysteine was used. The amino acid sequence used (mouse Insulin-A-chain: CSLYQLENYCN) is fully homologue to human insulin. A detailed description of the InsA autoimmunity model is provided in Amendt and Jumaa, 2021 (EMBOJ). Coupling of peptides to Streptavidin (SAV, ThermoScientific) was done by addition of biotin to the N-terminus. The C-terminus was left with an OH-group for better handling. 4-hydroxy-3-nitrophenylacetyl coupled to KLH (NP(30)KLH) or BSA (NP(15)BSA) were purchased from Biosearch Technologies.

Female C57BL/6, Esr1^−/− and Esr1^+/+ mice (8–12 weeks of age) were given water containing nil or VPA ad libitum 1 week before NP₁₆-CGG (average 16 molecules of 4-hydroxy-3-nitrophenyl acetyl coupled to 1 molecule of chicken γ-globulin; Biosearch Technologies) immunization until end of the experiments. For NP₁₆-CGG immunization, the mice were injected i.p. with 100 μg of NP₁₆-CGG in 100 μl of alum (Imject® Alum, Pierce). Serum was collected 10 days later for titration of circulating total and NP-binding IgM and IgG1 using enzyme-linked immunosorbent assays (ELISAs), as we described (27 (link), 39 (link)–41 (link)). Female Aicda^creEsr1^fl/fl and Aicda^creEsr1^+/+ mice were given plain water or SCFA-water containing 140 mM sodium butyrate and 20.0 mg/ml tributyrin starting at the age of 5 weeks and i.p injected with NP₁₆-CGG in 100 μl of alum at the age of 8 weeks. The mice were boost injected with NP₁₆-CGG in PBS 3 weeks later. Serum total and NP₄-binding IgM and IgG1 were measured by ELISA 7 days after boost injection.