Extended Data Figure 4. H3Q5ser and H3K4me3Q5ser antibody validations.

a, Synthesis of peptide antigens on 2-Cl trityl resin by (i) iterative Fmoc solid-phase peptide synthesis incorporating Fmoc-Glu(OAII)-OH at position 5 and either Fmoc-Lys(Boc)-OH or Fmoc-Lys(me₃)-OH at position 4, (ii) followed by Pd(0) deallylation, (iii) 5-HT coupling and (iv) acidolytic cleavage from the resin and global deprotection. Side-chain protecting groups are omitted for clarity. b, Peptide dot blot titrations testing the α-H3Q5ser antibody’s reactivity against unmodified vs. Q5ser peptides; note that linear signal was only observed with the Q5ser peptide. Direct blue (DB) staining was used to control for peptide loading. c, WB analysis of TGM2 serotonylation assays on unmodified mononucleosomes revealing that the α-H3Q5ser antibody only detects signal when the nucleosomes have been transamidated with 5-HT. DB staining was used to control for protein loading. d, Peptide competition WB analysis of lysates from RN46A-B14 cells indicating the specificity of our α-H3Q5ser antibody. e, Peptide dot blot titrations testing the α-H3K4me3Q5ser antibody’s reactivity against various peptides; note that linear signal was only observed with the K4me3Q5ser peptide. Direct blue (DB) staining was used to control for peptide loading. f, WB analysis of TGM2 serotonylation assays on monomeric K4me3 (EPL) H3 revealing that the α-H3K4me3Q5ser antibody only detects signal when the K4me3 histone has been transamidated with 5-HT. DB staining was used to control for protein loading. g, Peptide competition WB analysis of lysates from RN46A-B14 cells indicating the specificity of our α-H3K4me3Q5ser antibody in vivo; note that a minimum of 20 μg protein was loaded for all other comparisons made throughout the study. h, Peptide competition WB analysis of lysates from hPSC-derived 5-HT neurons indicating the specificity of our α-H3K4me3Q5ser antibody in human cells. For experiments in b-h, similar results were confirmed in ≥ 2 independent experiments/assay.