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. Author manuscript; available in PMC: 2019 Jan 22.
Published in final edited form as: Angew Chem Int Ed Engl. 2018 Jan 2;57(4):967–971. doi: 10.1002/anie.201706535

Figure 2.

Figure 2

Determine the labeling specificity of recombinant ST3Gal-IV in CHO cell mutants via western blot, flow cytometry, and imaging analysis. a) CHO cell mutants were treated with CMP-SiaNAl or CMP-Sia (500 μM) and ST3Gal-IV (50 μg mL−1) for 30 min. The labeled cells were then reacted with biotin-Az via CuAAC, lyzed, and probed with anti-biotin antibody (top). The Commassie blue stain proved equal loading amount (bottom). b) CHO cell mutants were biotinylated as described above. The labeled cells were probed with streptavidin-Alexa Fluor 488 for flow cytometry analysis (n = 3). c) Imaging of cell-surface glycans terminated with galactose. Lec2 cells stained with chloro-methyl fluorescein diacetate (CMFDA, red) and unstained Lec8 cells were mixed at a 1:4 ratio and cultured for 3 days. The cells were treated with ST3Gal-IV and CMP-Sia or CMP-SiaNAl, then stained with Alexa Fluor 647-Az (green) and Hoechst 33342 (blue). Scale bars: 20 μm.