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. 2020 Dec 17;8:622822. doi: 10.3389/fcell.2020.622822

FIGURE 2.

FIGURE 2

Imaging the actin cytoskeleton and actin-binding proteins within cells. (A) Zeiss Airyscan microscopy images of COS-7 cells probed with an actin-binding artificial non-antibody binding protein (“Affimer”) biotinylated on the C-terminal cysteine and visualized with fluorescent streptavidin (green). Cells were co-stained with fluorescent phalloidin (left panel; red) or antibody against non-muscle myosin 2B/MYH10 (right panel; red). Scale bars 10 or 5 μm, as indicated. (B) Confocal microscopy (upper panel) and high content imaging (lower panel) of ciliated serum-starved mouse inner medullary collecting duct (mIMCD3) cells expressing LifeAct-GFP (upper panel; green) or probed with AlexaFluor 488-phalloidin conjugate (lower panel; green). Primary cilia are marked by ARL13B (red). Frame indicates magnified inset showing detail of actin stress fibers in the upper panel. Scale bars = 20 μm. (C) Confocal microscopy image of human female dermal fibroblasts heterozygous for the FLNA frameshift mutation c.1587delG, p.(K529Nfs*40) (Adams et al., 2012) probed with antibodies for the actin-binding proteins filamin A (green) and filamin B (red), and counterstained with phalloidin-AlexaFluor633 conjugate (blue). The FLNA gene is carried on the X chromosome and random X-inactivation in different cells results in either haploinsufficiency (yellow-colored cell; indicated by arrow) or complete loss of filamin A protein (magenta-colored cell; arrowhead). Scale bar = 10 μm.