Yeast and human NTR1 interact with the respective orthologs of PinX1

Copyright information:

Taken from "Conserved interactions of the splicing factor Ntr1/Spp382 with proteins involved in DNA double-strand break repair and telomere metabolism"

Nucleic Acids Research 2007;35(7):2321-2332.

Published online 27 Mar 2007

PMCID:PMC1874655.

© 2007 The Author(s)

() Two-hybrid interaction of human NTR1 and PinX1. Two-hybrid analyses were performed with the indicated Gal-BD and -AD fusions of PinX1 and NTR1. Numbers in brackets indicate amino acids. Experimental conditions as in B. The interaction of LIG4 and XRCC4 (1–230) was used as a positive control. () Copurification of human NTR1 from bacteria expressing recombinant NTR1 and GST-fused PinX1. Experimental conditions as in D. western blot (WB) analysis of bacterial extracts and glutathione sepharose-bound proteins using anti-NTR1 antibody. Upper panel: expression of recombinant NTR1 in 20 μg of bacterial extracts in the presence of a GST-expressing vector (GST) and a vector expressing a GST-fused form of PinX1 (GST-PinX1). Lower panel: corresponding NTR1 copurification after pull down of GST-tagged proteins and several washes at high salt stringency. () Purification of PinX1 from HeLa cell extracts with GST-NTR1 (192–580). Experimental conditions as in C. Western blot analysis was performed with anti-PinX1. () Two-hybrid interactions of yeast Ntr1p and PinX1p. Experimental conditions as in B. () Overexpression of PinX1 mediates proximity between NTR1 and TRF1 in three-hybrid analyses. Serial dilutions of two randomly picked clones. Experimental conditions as in . TRF1 and NTR1 (1–580) were fused to the Gal4-BD and -AD, respectively. PinX1, XRCC4 or NTR1 were expressed under the control of a methionine-repressible promotor whereever indicated.