Degree of Eph clustering determines receptor activation. (A) Representative blue native PAGE blot for autophosphorylation analysis of single EphB2 oligomeric species (B–D). Blue native PAGE of lysates of COS-7 cells expressing different FKBP isoforms of wtEphB2 and stimulated with either vehicle (−) or AP20187 (250 nM for 20 min). Western blot was performed with anti-phospho-EphB2 antibodies; blots were stripped and reblotted for total EphB2 protein (see Fig. S3 D). The relative phosphorylation levels of single oligomeric species were measured according to example regions outlined by dashed boxes for monomers, dimers, and species greater than or equal to trimers. (B) Quantification of the cumulative relative phosphorylation per lane displayed as mean ratio ± SEM of phosphorylated versus total EphB2 protein over n = 4 blue native PAGE experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA with post hoc Bonferroni test; asterisk in red represents significance level to 1FKBP control stimulation). (C) Quantitative autophosphorylation analysis of single cluster species displayed as mean ratio ± SEM of phosphorylated versus total EphB2 protein from example regions in A from n = 4 blue native PAGE experiments normalized to dimer phosphorylation level set to 1.0 (*, P < 0.05, one-way ANOVA with post hoc Bonferroni test). (D) Relative abundance of cluster species in percentage ± SEM of cumulated species population (*, P < 0.05; ***, P < 0.001; Student’s t test). Data are derived from optical density quantification of regions outlined by dashed boxes in A on Western blots for total EphB2 (see Fig. S3 D) of n = 4 blue native PAGE experiments.