Fig. 2.

Identification of human augmin as the 8-subunit complex. (A) Immunoblotting of hDgt6 for the control, siRNA-treated, and GFP-hDgt6-expressing cells. GFP-hDgt6 was expressed 2-fold more than endogenous hDgt6. (B) Coimmunoprecipitation of 7 HA-tagged hDgt6-associated proteins with endogenous hDgt6. The supernatant (S) and precipitated (P: 50× volume of S) fractions after anti-HA incubation were immunoblotted with ati-hDgt6 antibody. The asterisks in A and B indicate cross-reaction of anti-hDgt6 antibody to other proteins. (C) Localization of GFP-tagged C14orf94 and C4orf15 proteins (green) in metaphase (Left) and interphase (Right). The proteins were abundant at centrosomes during interphase, whereas spindle localization with slight enrichment near the poles was detected during metaphase. Chromosomes (blue) and γ-tubulin (red) were counterstained. (Scale bar, 5 μm.) (D) Two-hybrid interaction between full-length Hice1 and hDgt6. Colonies were visible on the 3-aminotriazole (3-AT)-containing plate only when both proteins were expressed. (E) Gel filtration analysis of human augmin subunits. A similar peak can be observed between hDgt6 and the hDgt6-interacting proteins at a Stokes radius of ≈9.5 nm, suggesting that they form a stable complex. The peak of KIAA0841 was shifted by one fraction to the left, probably because of GFP tagging of this protein (others were tagged with HA). The reason for the shift of Hice1 to higher molecular mass fractions is unclear but might be aggregation of a subpopulation of this protein in the extracts.