Fig. 1: PFKFB3 is modified by O-GlcNAc.
From: O-GlcNAcylation of PFKFB3 is required for tumor cell proliferation under hypoxia
a, b SW1990 cells (a) with Flag-PFKFB3 and OGT shRNA expression (b) were cultured for 12 h under hypoxia or normoxia. The O-GlcNAc modified proteins modified by azide were labeled with biotin and isolated with streptavidin beads for immunoblotting analyses. c Flag-PFKFB3 was expressed in SW1990 cells. Immunoprecipitation analysis was performed using the anti-Flag antibody, and the extracts were analyzed by mass spectrometry. Precursor mass shift with HexNAc modification, measured with high mass tolerance (5 ppm); existence of signature HexNAc+1 fragment ions in MSMS spectra; existence of site localization ions (y19+) that covers the modified S172; almost complete y ion series for the peptide (Carb stands for carbamidomethyl). These evidences indicate that S172 was O-GlcNac modified. d, e SW1990 cells with indicated WT or mutant Flag-PFKFB3 (d) or SW1990 and HPDE cells with indicated glucose concentrations (e) were cultured for 12 h under hypoxia or normoxia. The O-GlcNAc modified proteins modified by azide were labeled with biotin and isolated with streptavidin beads for immunoblotting analyses. In a, b, d and e, immunoblotting analyses were performed using the indicated antibodies.
