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Fig. 9. The roles of the actin and the microtubule cytoskeletons, and Nak1/Orb3 kinase activity in localizing the protein. (A-C) The effects of F-actin and microtubule depolymerization on the localization of Nak1/Orb3-GFP. Cells were incubated with 10 µM latrunculin A for 20 minutes (+ Lat A), with 25 µg ml–1 of the microtubule-depolymerizing agent carbendazim (+ MBC) or with their solvent dimethylsulfoxide as a control (+ DMSO). Arrows show patches of Nak1/Orb3-GFP. Samples with and without MBC were fixed in methanol and stained with anti-tubulin antibodies to control for microtubule polymerization (data not shown). (D) Effects of nak1/orb3-167 mutation on Nak1/Orb3-GFP localization. Nak1/Orb3-GFP cells grown at 25°C and cells shifted to 36°C for 60 minutes were fixed in formaldehyde and stained with anti-GFP antibodies. Bars, 10 µm.





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