CAPNb2 blocks CapG interaction with monomeric actin and actin filaments. (A) G-actin-binding assay. CAPNb2 and 3 counteract a CapG-triggered increase in pyrene-labeled actin fluorescence. Intrinsic fluorescence of pyrene-actin was normalized to 1. A, actin; C, CapG; RFUs, relative fluorescence units. (B) Principle of the actin sedimentation assay. Actin monomers freely polymerize in the absence of CapG (1). CapG sequesters actin monomers, preventing their incorporation into filaments (2). If a nanobody prevents interaction between CapG and actin monomers, the sequestration effect is lost, and actin will polymerize (3). A nanobody may interact with CapG without affecting the sequestration of actin monomers, resulting in decreased polymerization (4). (C) CAPNb2 reduces the ability of CapG to sequester actin monomers in a sedimentation assay. Supernatants (SNs) and actin pellets (Ps) were fractionated with high-speed centrifugation. Proteins were separated with SDS-PAGE and visualized with Coomassie staining. Lanes 1, 2: controls. BSA does not affect actin polymerization. DNaseI (nanomolar affinity for G-actin) strongly blocks actin polymerization. Lanes 3/, –4: CapG reduces actin sedimentation. Actin level in the pellet is reduced while actin in the SN is increased. Lane 5: CAPNb2 counteracts the effect of CapG by increasing the actin fraction in the pellet and decreasing actin in the SN. Lanes 6 through 9: Other nanobodies have no significant effect. (D) Quantification of the experiment in (C). A, actin; C, CapG. (E) F-actin-capping assay. Capping of F-actin seeds by CapG prevents the initial increase in fluorescence in the control condition and an increase in final F-actin level. The initial increase is due to high elongation rates of added F-actin seeds. CAPNb2 prevents CapG-dependent F-actin capping and results in actin polymerization kinetics comparable to the control. Student t test was performed: *P < 0.05; **P < 0.005; n = 3.