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. 2022 Aug 18;298(9):102400. doi: 10.1016/j.jbc.2022.102400

Figure 1.

Figure 1

In vitro and intracellular activity of GnT-IVa and GnT-IVb.A, schematic model of the GlcNAc transfer reaction catalyzed by GnT-IVa and GnT-IVb (MGAT4A and MGAT4B). B, plasmid constructs used in this study. C, FACS analysis of HEK293 WT (blue), HEK293 GnT-IVa KO (red), and HEK293 DKO cells (orange) with DSA lectin (left). The right graph shows the geometric means (n = 3, means ± SD, ∗∗∗∗p < 0.0001, Tukey's multiple comparisons test). D, the endogenous GnT-IV activity in HEK293 WT, GnT-IVa KO, and DKO cells was measured by incubating the lysates with the PA-labeled acceptor sugar (GnGnbi-PA) and analyzing by HPLC. E, the specific activity of GnT-IV in the HEK293 WT, GnT-IVa KO, and DKO cell lysates calculated by the peak area in (D) (n = 3, means ± SD, ∗∗∗∗p < 0.0001, Tukey's multiple comparisons test). F, soluble GnT-IVa and GnT-IVb were expressed in COS7 cells and purified from the media using a Ni2+ column. Purified GnT-IVa and GnT-IVb were separated by SDS-PAGE and visualized by CBB staining. G, enzyme activity of the purified GnT-IVa and GnT-IVb was measured by incubating the enzymes with GnGnbi-PA and analyzing by HPLC. H, the specific activity of the purified GnT-IVa and GnT-IVb was calculated by the peak area in (G) (n = 3, means ± SD, ∗∗p < 0.01, unpaired t test). I, proteins from mock-treated HEK293 WT cells and DKO cells transfected with an empty vector (mock) or a plasmid for expression of GnT-IVa or GnT-IVb were subjected to SDS-PAGE and blotted with anti-myc antibody (upper left), anti-GAPDH antibody (lower left), or HRP-conjugated DSA (right). J, lysates of mock-treated HEK293 WT cells and DKO cells transfected with an empty vector (mock) or a plasmid for expression of GnT-IVa or GnT-IVb were reacted with GnGnbi-PA and analyzed by HPLC (n = 3, means ± SD, ∗∗∗∗p < 0.0001, Tukey's multiple comparisons test). CBB, Coomassie brilliant blue; DKO, double KO; DSA, Datura stramonium agglutinin; FACS, fluorescence-activated cell sorting; GnT, N-acetylglucosaminyltransferase; HEK293, human embryonic kidney 293 cell line; HRP, horseradish peroxidase.