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. 2020 Jun 23;6:42. doi: 10.1038/s41421-020-0170-2

Fig. 2. Direct binding to Ras positions mitotic Cdc24.

Fig. 2

a Two-hybrid. EGY48 cells, carrying LacZ under control of LexAop, were transformed with the indicated bait and prey constructs (described in “Materials and methods” section). Isolated transformants were patched on selective plates containing raffinose, galactose, to induce the preys, and X-Gal to detect β-gal activity; blue staining is indicative of physical interaction between fusion proteins. b Cells of given strains were arrested in nocodazole. Immunoprecipitation of Cdc24-GFP was conducted as described in “Materials and methods” section and immunoblot analysis with antibodies against GFP or Ras2 performed. The plot in c represents the percentage of cells with polarized GFP-Cdc24 in the indicated strains after 3 h of nocodazole treatment. No substantial differences in GFP-Cdc24 expression levels were detected (Supplementary Fig. S2b). Five (panel c) independent experiments were performed, counting 100 cells for each repeat. Green, magenta, and cyan arrows show cells with even PM Cdc24 distribution, sites of polarized GEF and cells with cytoplasmic Cdc24, respectively. Scale bars in c: 5 μm. t-test was applied as a statistical measurement in c; n.s.: not significant; *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.