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. 2018 Jan 12;8:612. doi: 10.1038/s41598-017-18765-1

Figure 5.

Figure 5

(a) Structure of GFP-tagged Y14 mutants that lack MAGOH binding ability. (b) Expression of the transfected GFP-Y14 L118R mutant was detected by western blotting with an anti-GFP antibody (left panel). Phosphorylation status of GFP-Y14 L118R was analyzed by separation using phos-tag gels (right panel). The samples for SDS-PAGE and phos-tag SDS-PAGE derive from the same experiment. The membranes were cut prior to exposure so that only the portion of gel containing the desired bands would be visualized. Full-length blots are shown in Supplementary Fig. S10. (c) Expression of the transfected GFP-Y14 ΔRRM mutant was detected by western blotting with an anti-GFP antibody (left panel). Phosphorylation status of GFP-Y14 ΔRRM was analyzed by separation using phos-tag gels by comparing with phosphatase-treated GFP-Y14 ΔRRM (right panel). The cropped blots are used in the figure. The samples for SDS-PAGE and phos-tag SDS-PAGE derive from the same experiment. The membranes were cut prior to exposure so that only the portion of gel containing desired bands would be visualized. Full-length blots are shown in Supplementary Fig. S10. (d) Localization of GFP-Y14, L118R, and ΔRRM mutants was observed by fluorescence microscopy (green). Fibrillarin and nuclear DNA are shown as red and blue, respectively. Bar indicates 20 μm. (e) GFP-Y14 and CSA mutant were observed by fluorescence microscopy (green). Localization of Flag-MAGOH and nuclear DNA is shown as red and blue, respectively. Structures of mutants are shown in Fig. 2. Bar indicates 20 μm.