Human EXT1 expressed with mouse or bovine EXT2 in BHK or mouse sog9 cells forms associated complexes. Figure 2 shows Golgi redistribution, Figure 3 shows tagged self/hetero-association, and Table 1 assays cellular immunoprecipitates rather than partner-free EXT1.

Human soluble EXT1 expressed separately in HEK293 cells has positive single-donor transfer and chain-extension activity on K5-derived acceptors (single-transfer Results/Figure 5); Figure 6 shows an apparent 160-kDa EXT1 species and coeluting tagged EXT1–EXT2. The endogenous-partner IEF check is reported as data not shown.

Human-targeted EXT1 siRNA in HEK293 cells shortens HS chains and lowers the HS/chondroitin-sulfate ratio (silencing Results/Figure 2). The separate full-length overexpression constructs are mouse-brain cDNAs, and the Figure 6 localization experiment concerns EXT2.

The purified human EXT1–EXT2 complex is a 1:1 heterodimer. EXT1 GT-B mutations impair GlcA transfer; both GT-A mutant sets affect GlcNAc transfer in these assays, with altered stability of the EXT1 GT-A mutant preparation limiting a purely catalytic interpretation.

Purified human EXT1–EXT2 assigns robust GlcA transfer to EXT1 and dominant GlcNAc transfer to EXT2. Low-yield, proteolyzed EXT1-only material nevertheless shows low qualitative activity; these results do not establish universal EXT1 GlcNAc inactivity.

Human HeLa-derived EXT1 expressed in mouse/CHO hosts restores or alters cell-surface HS. Tagged EXT1 has an ER pattern; deleting its first 26 residues disrupts that pattern and HSV-based functional rescue (Figures 3 and 5). The type-II topology diagram is a model, not a protease-protection measurement.