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. 2020 May 31;12(6):1430. doi: 10.3390/cancers12061430

Figure 3.

Figure 3

In vitro O-fucosyltransferase assay for activity of WT and mutated POFUT1 variants using click chemistry. (a) After 1 h or 20 h incubation at 37 °C of recombinant WT human POFUT1 with NOTCH1 EGF-LD 26 (WT and T997A) or EGF-LD 12 (WT and T466A) and GDP-azido-fucose, click chemistry (CuAAC) with alkynyl biotin was performed to link biotin to transferred O-linked fucose covalently. After SDS-PAGE and blotting techniques, EGF-LDs modified with O-fucose were revealed using HRP-streptavidin. (b) After 1 h independent incubations of WT POFUT1 and each mutated POFUT1 variant with WT or T997A EGF-LD 26 at 37 °C, the same procedure as described in (a) was carried out. A low exposure did not allow us to visualize WT POFUT1 labelling. After high exposure, the signal for WT POFUT1 was detected, and quantifications were performed for each POFUT1 variant compared to WT. Uncropped blots are shown in Figures S2 and S3.