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. 2014 Oct 20;111(44):15723–15728. doi: 10.1073/pnas.1417993111

Fig. 2.

Fig. 2.

Xylosyl kinase Fam20B globally regulates glycosaminoglycan chain elongation. (A, Top) A 3G10 immunoblot of U2OS cell surface proteoglycan heparan sulfate GAG chains from FAM20B wild type (WT) and two different FAM20B TALEN knockout (KO) clones, with or without heparin lyase (hepase) and Chon-ABC treatment. (Middle two panels) Immunoblots of endogeneous Syndecan 1 (SDC1) and Glypican 1 (GPC1) from FAM20B WT and KO cells, with or without hepase and Chon-ABC treatment. (Bottom two panels) Knockout of FAM20B was demonstrated by DNA sequencing and anti-Fam20B immunoblotting, and GAPDH served as a loading control. The electrophoretic mobilities of SDC1 and GPC1 on the 3G10 blot are marked by an asterisk and arrowhead, respectively. (B) Quantification of cell surface SDC1 was carried out in the WT and KO cells using flow cytometry with primary anti-SDC1 antibody and PE-labeled secondary antibody. The binding of secondary antibody alone to the cell surface served as a background control measurement. (C) Immunoblotting of SDC1 was carried out in intact FAM20B WT and KO cells following treatment with or without trypsin. (D) Schematic representation of Fam20B function on proteoglycan biosynthesis and sorting. Blue lines represent mature GAG chains and red lines represent short sugar stubs that remain on the core protein in FAM20B KO cells.