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Fig. 7 | BMC Biology

Fig. 7

From: Bex1 is essential for ciliogenesis and harbours biomolecular condensate-forming capacity

Fig. 7

Bex1 reconstitutes tubulin polymerization. a Image derived from unfixed cells expressing EGFP-Bex1. EGFP-Bex1 forms cytoplasmic granules. Scale bar = 5 μm. b Immunostaining for α-tubulin in the cells expressing EGFP-Bex1. The EGFP-Bex1 granules accumulate α-tubulin. Scale bar = 5 μm. c The 2D-NMR spectroscopy with 15N-labelled recombinant Bex1 protein in the presence of indicated molar ratios of Bex1 to guanosine triphosphate (GTP). The 1H-15N HSQC spectra were measured at pH 7.3 and 25°C. The increased GTP provoked the peak shifts (indicated by arrowheads) in a dose-dependent manner. d Calculation of the dissociation constant (Kd) for Bex1 and GTP. The distance of the peak shifts (Δδ) obtained in (C) was plotted as the filled dots to calculate the dissociation constant of Bex1 and GTP. For comparison, the plots from the upper right peak that did not shift were shown as the open dots. e Concentrations of reagents for the analysis of the Bex1 condensate formation. f Formation of Bex1 condensates in the presence or absence of PEG4000. Recombinant Bex1 protein was labelled with DyLight fluorescent dye. Scale bar = 5 μm. g The magnified view of the Bex1 condensate. Scale bar = 1 μm. h Concentrations of reagents for the analysis of tubulin polymerization in the presence of the Bex1 condensates. i Representative images of polymerized tubulin (magenta) provoked by the presence of the Bex1 condensates (green). Tubulin protein was labelled with Alexa fluorescent dye. Scale bar = 1 μm. j Histogram of the length (μm) of the polymerized tubulin in the presence of the Bex1 condensates. k Length (μm) of the polymerized tubulin. Without recombinant Bex1 protein, no tubulin polymerization was observed. l Schematic representation of the cell density-dependent role of BEX1 in regulating cellular homeostasis. At a low cell density, BEX1 localizes to nucleoli, where BEX1 contributes to the formation of DFCs. At a high cell density, BEX1 localizes to the apical side of cells and contributes to the formation of apical microtubules and primary cilia. p < 0.001; Student’s t test. The data are presented as the means ± standard deviations

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