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. 2007 Dec 28;3(12):e234. doi: 10.1371/journal.pgen.0030234

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© 2007 Al-Ramahi et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) GST Co-AP pull-down experiments between dAtx2 and GST-constructs carrying human Ataxin-1[82Q] or Ataxin-1[82Q] with S776A mutation.

(B) GST Co-AP experiments between human Ataxin-2 (Myc-hATXN2) and GST-Ataxin-1 with different polyglutamine lengths.

(C) Interaction between GST-Ataxin-1[2Q] and endogenous hATXN2 in cell culture.

(D) Co-AP pull down experiments between GST-Ataxin-1 and Flag-dAtx2 after nuclear/cytoplasmic fractionation. Tubulin is used as cytoplasmic marker and Mecp2 as nuclear marker.

(E) Comparative analysis of the interaction of hAtaxin-2 with different domains of the Ataxin-1 protein. Lane-1, GST alone does not pull down Myc-hAtaxin2. Lane-2, GST-ATXN1^82Q pulls down Myc-hAtaxin2. Lane-3 expanded N-terminal Ataxin-1 (GST-ATXN1N-term^82Q) lacking the AXH domain (aa# 1–575) also pulls down Myc-hAtaxin-2 with high affinity. Lanes-4 and 5 show that both the C-terminus portion of Ataxin-1 containing the AXH domain (aa# 529–816; lane-4) or the AXH domain alone (aa# 558–700; lane-5) pull down Myc-hAtaxin2. These interactions are weaker than that of the N-terminus part (compare lanes 4 and 5 with lane 3). Cell lysates for co-AP were ran through Glutathione conjugated beads, and blots stained with anti-GST or anti-Myc. Immunoblots for dAtx2, Myc-hAtaxin2 and GFP carried out on cell lysates before the co-AP pull-down reveal similar levels of both proteins between samples.