The actin-bundling activity of AnxA2 is linked to the formation of GM1-enriched domains in stimulated cells. (A) F-actin binding assay of recombinant GST-AnxA2 WT and GST-AnxA2 K286A in the absence and presence of calcium. Purified recombinant AnxA2 proteins fused with GST (4 µM) were incubated with preformed actin filaments (18 µM) for 30 min at room temperature. After low-speed centrifugation, the supernatant (S) and pellet (P) were collected and separated on a 4–20% SDS-PAGE-gel. (B) Electron microscopic visualization of recombinant AnxA2/F-actin aggregates. Before centrifugation, a 5-µl aliquot was spread on electron grids and prepared for electron microscopy. The inset shows a higher magnification of the region delimited by the square. (C) Cells expressing AnxA2-WT-GFP or AnxA2-K286A-GFP were stimulated with 59 mM K⁺ in the presence of fluorescent cholera toxin to visualize GM1-enriched domains, then fixed and stained with TRITC-phalloidin. Confocal images were recorded in the same optical section. Asterisks indicate nontransfected cells. (D) Semiquantitative analysis of cholera toxin (GM1) and F-actin labeling in nicotine-stimulated cells expressed in arbitrary units (±SEM; n = 20). Statistical significance for medians (black line) was determined using a Mann-Whitney test. Asterisks indicate statistical significance (*, P < 0.05; ***, P < 0.001) and the white lines represent the means. Similar results were obtained on three culture preparations. Bars: (B, main panels) 500 nm; (B, inset) 100 nm; (C) 10 µm.