MX segment binds RIC-3. (a) Sequence alignment of L1-MX– and MX-peptides with a partial 5-HT_3A sequence (top) consisting of the last 13 amino acids of M3 (green), L1-segment (blue), MX-helix (magenta) and 36 residues of the long L2-loop (blue) that connects with the MA-helix (blue, not shown in a, only in b). L1-MX-C contains a C-terminal Cys, and L1-MX-N and MX contain an N-terminal Cys. (b) Ribbon representation of a single 5-HT_3A subunit indicating L1 and MX and other named peptide segments within the transmembrane and intracellular domains (PDB ID 4PIR; Hassaine et al., 2014). (c and d) Eluates of resin-linked peptide pull-downs of RIC-3. Western blot images show RIC-3 expressed in oocytes and control (no RIC-3 injection). Eluates from resin alone show the presence of a minor amount of RIC-3, whereas eluates from resin covalently linked with L1-MX-N peptide show prominent RIC-3 bands. 1×, 2×, and 4× indicate increasing peptide to resin ratios. Eluates from four independent pull-down experiments are presented in lane #6 of c, L1-MX-N, and in lanes #3, 4, and 5 of d, 1× L1-MX-N, 2× L1-MX-N, and 4× L1-MX-N; data shown here are technical replicates that used the same batch of L1-MX-N peptide and RIC-3 extracted from the same batch of n = 84 Xenopus oocytes. For biological replicates of L1-MX and RIC-3 assay, n ≥ 5 were obtained, which used RIC-3 from five different batches of Xenopus oocytes and three different batches of L1-MX peptides purchased from two different companies. (e) Eluate from MX-linked resin yields RIC-3 band; data shown here is the representative of n = 3 biological replicates, where RIC-3 was extracted from three different batches of Xenopus oocytes. Source data are available for this figure: SourceData F2.