Figure 2.
Both N- and O-glycans Serve as SubAB Receptors
(A) SubAB sensitivity in glycan-gene KO cells. Cells were treated with SubAB as described in Figure 1B at the indicated concentration. Viability was estimated as described for Figure 1B and is expressed as the mean percentage of the MTT value (OD570) in the absence of SubAB. The percentages shown are the mean percentages ± SD obtained from three independent experiments.
(B) BiP cleavage in glycan-gene KO cells. Cells were incubated with SubAB at the indicated concentration of SubAB for 12 h. SubAB-induced BiP cleavage was determined by immunoblots using anti-BiP monoclonal antibodies. GAPDH served as a loading control. Quantification of BiP uncleaved (78 kDa) by SubAB was performed by densitometry. The percentages shown are the mean percentages ± SD obtained from three independent experiments. The Bonferroni-corrected t test was used for multiple comparisons. *p < 0.017.
(C) Surface binding of SubAB on glycan-gene KO cells. Cells were stained with (blue lines) or without (black lines) Alexa 488-labeled SubAB (Alexa 488-SubAB) and analyzed using FACS. Yellow-green lines in all panels indicate staining in parent cells.
(D) Detection of SubAB-binding proteins in glycan gene KO cells. Biotinylated cell surface proteins prepared from the indicated cells were immunoprecipitated with heat-inactivated (HI) or native (N) SubAB as described in the Transparent Methods section. SubAB-binding proteins were detected with streptavidin-horseradish peroxidase (HRP). Data are representative of at least three separate experiments.