Fig 1: Binding of half-processed Activin-A to type II activin receptors, and analysis of its βA subunits on reducing gels and by mass spectrometry.A Western blot of representative B16F1-mS1 and FurKO#2-ctrl or -βA cell SNs, and ImageJ quantification of the proportions of mature, hemicleaved, and uncleaved Activin-A in independent sample preparations from the indicated cell lines used as inputs to test Activin-A binding to immobilized ActRIIB-Fc by plasmon resonance measurements. The microfluidic chamber used to inject cell SNs on a nanoplasmonic chip functionalized with ActRIIB-Fc on gold nanoparticles is depicted on the right. Drops in signal intensity are induced by Activin-A binding to ActRIIB-Fc. B Binding of the different forms of Activin-A was evaluated by injecting the B16F1-βA or FurKO#2-βA cell SNs analyzed in (A). B16F1-mS1, or B16F1-Ctrl cells expressing empty lentivirus served as specificity controls. Error bars, SEM (n = 3 independent experiments); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t-test. C Schematic of the conversion of hemicleaved Activin-A (A70) to A60 by furin cleavage of the second βA subunit, and strategy to monitor the cleavage using anti-Flag and anti-Activin-A (ActA) antibodies. In this model, an allosteric cystine bond (S–S) links the prodomain (violet) of one βA subunit to the C-terminal region (yellow) in A70 and A60. Prodomain that is not covalently attached (light shading) can be displaced by cognate type II receptors. D Anti-Activin-A (top) or anti-Flag Western blots (bottom) of A70 in SN of FurKO#1 cells expressing Flag-tagged 27F-βA analyzed before (input) and after pull-down by AIIB-Fc. SN of B16-F1 cells, and of mock-transfected FurKO#1 cells served as controls. Arrow, cleaved prodomain; n.s. non-specific. E Anti-Activin-A (top) or anti-Flag (bottom) Western blot of the indicated Flag-tagged Activin-A constructs after pull-down as in (D), but on reducing gels. FurKO#1 and B16-F1 control SNs used to pull down Flag-tagged A70 and A60 encoded by 130F-βA or 146-βA were as in Fig. S3C. F LC–MS analysis and spectral counts of peptides mapping to Activin-A in samples collected in (E). Data represent two pull-down experiments, one using ActRIIA-Fc and the other using AIIB-Fc. Schemas were created using BioRender.com.