Recombinant Llama Anti-GSN Single Domain Antibody (HPAB-0342CQ) (CAT#: HPAB-0342CQ)

Figure 1 Gelsolin single-domain antibodies (GsnVHHs) recognize different gelsolin populations in cells.

SDS-PAGE Coomassie staining of purified recombinant VHHs. GFP VHH is a green fluorescent protein sdAb used as a negative control in subsequent experiments. b His₆-tagged VHHs detect overexpressed and endog enous gelsolin similar to a commercial antibody. Cytoplasmic extracts of MCF-7 cells, HEK293T cells, gelsolin overexpressing HEK293T cells (Hek + pWPI gelsolin) or CapG overexpressing HEK293T cells (Hek + pWPI CapG) (30 μg protein) were electrophoresed and Western blotted with 4 μg His-tagged gelsolin VHH (left panel) or commercial antibody (right panel). Hek cells express gelsolin but the amount is below the detection limit. c Selected GsnVHHs expressed as intrabodies immunoprecipitate endogenous gelsolin. Left expres sion of Flag-tagged GsnVHHs following lentiviral transduction in MDA-MB-231 cells. Right immunoprecipitation of gelsolin from MDA-MB-231 cells using anti-Flag IgG-sepharose and mAb to gelsolin. The lysis buffer did not contain EGTA. d GsnVHH 11 and 13 recognize distinct gelsolin conformations in cells. SDS-PAGE of immunoprecipitates from lentiviral transduced MDA-MB-231 cells. In the absence of EGTA, GsnVHH 13 binds a gelsolin:actinn complex; GsnVHH 11 binds to free gelsolin (left panels). In the presence of EGTA, GsnVHH 13 does not bind gelsolin whereas GsnVHH 11 binds free gelsolin (right panels). *IgG heavy chain; **IgG light chain. Protein size markers are indicated at left. e Western blots showing the presence of gelsolin (upper panel), actin (middle panel) and VHH (bottom panel) in immunoprecipitates as in (d), either without EGTA in the lysis buffer (left) or with EGTA in the lysis buffer (right)

Van den Abbeele, A., De Clercq, S., De Ganck, A., De Corte, V., Van Loo, B., Soror, S. H., ... & Gettemans, J. (2010). A llama-derived gelsolin single-domain antibody blocks gelsolin–G-actin interaction. Cellular and molecular life sciences, 67(9), 1519-1535.

Figure 2 GsnVHHs bind to distinct domains in the amino- or carboxyterminal half of gelsolin.

a Pull-down experiment showing binding of GsnVHH 13 to GST-full-length gelsolin (lane 2), GST-G4-G6 (lane 4) and GST-G4-G5 (lane 8) but not to other gelsolin domains. Asterisks indicate full-length fusion proteins and the dots mark VHHs. b Schematic overview of pull-down experiments. GsnVHHs 7-11 bind to the amino-terminal half of gelsolin (G1-G2 or G2-G3). GsnVHHs 3, 6, 12, and 13 bind to the carboxy-terminal half of gelsolin (G4-G5)

Van den Abbeele, A., De Clercq, S., De Ganck, A., De Corte, V., Van Loo, B., Soror, S. H., ... & Gettemans, J. (2010). A llama-derived gelsolin single-domain antibody blocks gelsolin–G-actin interaction. Cellular and molecular life sciences, 67(9), 1519-1535.

Figure 3 Selected GsnVHHs protect against gelsolin proteolysis.

SDS-PAGE Coomassie stained gels showing proteolysis of gelsolin over time (2, 5, and 8 days) in the presence of 100 lM Ca²⁺. GsnVHHs 2, 4, 6, and 11 accelerate gelsolin degradation in comparison to the control (c, control). GsnVHH 3 and 9 slow down degradation (asterisks).

Van den Abbeele, A., De Clercq, S., De Ganck, A., De Corte, V., Van Loo, B., Soror, S. H., ... & Gettemans, J. (2010). A llama-derived gelsolin single-domain antibody blocks gelsolin–G-actin interaction. Cellular and molecular life sciences, 67(9), 1519-1535.