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. Author manuscript; available in PMC: 2018 Jan 1.
Published in final edited form as: FEBS Lett. 2017 Jan 1;591(1):47–55. doi: 10.1002/1873-3468.12525

Fig. 2. Replacement of human spacer-1 with a GGS linker results in cleavage at Q882.

Fig. 2

A. Schematic of GlcNAc-1-PT α/β subunit modular arrangement and replacement of the human spacer-1 sequence with a 26 aa linker sequence comprising of Gly and Ser residues. B. Immunoblot analysis of WT α/β versus the DS1 and ΔS1 deletion mutants expressed in GNPTAB−/− HeLa cells and probed with anti-V5 antibody. C. Phosphotransferase activity toward the simple sugar αMM, using extracts of GNPTAB−/− cells transfected with WT α/β precursor or the ΔS1 mutant cDNA. Activity was normalized to total protein concentration. D. Inhibition of S1P activity of GNPTAB−/− HeLa cells transfected with either WT α/β precursor, the DS1 or the ΔS1 mutant cDNA. 24 h post-transfection, PF-429242 was added to the cells at a final concentration of 10 µM and cells were incubated for a further 24 h before being harvested. Cells extracts were prepared and 20 µg of each lysate was separated by SDS-PAGE and subject to western blotting.