MeCP2-TBLR1 Binding Assay Protocol

Western blotting was performed using antibodies against NanoLuc (Promega), MeCP2 (Sigma, M6818) and Sin3A (Abcam, Ab3479). The 35 amino acid MeCP2 wild-type and R306C NID peptides (residues 285-319) were the same as described previously^{25 (link)} and were used at a concentration of 20 µM in the competition assays. The biotin at the N-terminus of these peptides was not relevant to the experiments in this work. The SPOT peptide array had peptides based on mouse MeCP2 residues 297–308 (HETVLPIKKRKT). Residues 302–305 (PIKK) – which make direct contact with TBLR1—were systematically altered to each of the 20 naturally occurring amino acids^{49 (link)}. The array membrane was incubated with recombinant HIS₆‐tagged TBLR1 (2 µM) and immunodetection was performed with anti-HIS antibody (Sigma, H1029). Uniform synthesis efficiency across the array was ascertained by UV absorption. The higher affinity TBL1/TBLR1 binding mutant (K304Y) was incorporated into a 12 amino acid peptide (Ac-ETVLPIYKRKTR-NH₂) corresponding to residues 298-309 of MeCP2 which was designated UMT026-2. Note the shorter N-terminus and longer C-terminus of this peptide compared to the sequences used on the SPOT array. UMT026-2 was used at 50 µM when calculating the Z-factor^{39 (link)}.