Fig 1: Experimental validation of PepMLM-designed peptide binders in vitro.Four NCAM1-targeting peptides were designed for both RFdiffusion and PepMLM as C-terminal SUMO-tag fusions and screened for potential binding to NCAM1 (Supplementary Fig. 8). a, Binding analysis of top candidate peptide against NCAM1 fused to C-terminal SUMO-tag as measured by ELISA. PepMLM-designed peptides were immobilized onto 96-well plates, incubated with serial dilutions of either NCAM1-Fc or BSA and subsequently detected with an anti-IgG–HRP conjugate and the signal was quantified by measuring the absorbance at 450 nm (A450). Data are represented as the mean ± s.e.m. (n = 3 biological replicates). b, Further ELISA binding analysis of pMLM_NCAM1_2 comparing successive serial dilutions of NCAM1-Fc to BSA control (200 nM) (n = 12 biological replicates) (P = 0.0051, P = 0.003, P < 0.0001, P < 0.0001). Box plots represent the mean value bounded by the IQR, with brackets representing minimum and maximum values. (Statistical significance was determined by an ordinary one-way ANOVA followed by a Dunnett’s multiple comparison test. Calculated P values are represented as follows: **P < 0.01; ***P < 0.001; ****P < 0.0001.).