Ntr1p interacts with Lif1p

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)

() Amino acid alignment of the conserved core region of Lif1p and human XRCC4. () Mapping of Ntr1p and Dnl4p interaction domains of Lif1p. Left panel: β-Galactosidase assays of yeast two-hybrid analyses; right panel: two-hybrid analyses with serial dilutions of two independent colonies plated on non-selective (-Leu-Trp) and selective (-Leu-Trp-His) media. Different fragments of Lif1p were expressed as BD (Gal4 DNA-binding domain) fusion proteins (numbers in brackets indicate amino acids). Gal4 activation domain (AD) constructs are fusions of the coding sequences for amino acids 89–509 of Ntr1p and the entire ORF of Dnl4p. LTA is SV40 large T-antigen, pLAM5 is human lamin C (66–230). () Mapping of Lif1p interaction domain of Ntr1p with the indicated fusion proteins of Lif1p and Ntr1p. Two-hybrid analyses were performed as described for the right panel of B. () Copurification of Lif1p from bacteria expressing recombinant Lif1p and GST-fused Ntr1p. Western blot (WB) analysis of bacterial extracts and glutathione sepharose-bound proteins using anti-Lif1p antibody. Upper panel: 20 μg of extracts expressing Lif1p and GST-Ntr1p proteins as indicated. Lower panel: Lif1p copurification from 1 mg of soluble proteins after pull down of GST-tagged proteins and several washes at high salt stringency. Co indicates the vector control for Lif1p. Here, 5 µl out of 30 µl of proteins eluted from the beads were applied. () Copurification of Lif1p from expressing 6× histidine-fused Ntr1p (His-Ntr1). Co indicates the vector control. Proteins were co-expressed in yeast and histidine-fused proteins were purified using nickel agarose. Amounts of proteins used for affinity purification and WB analysis were the same as in (D). WB analysis of relevant elution fractions using anti-Lif1p or an antibody directed against 6× histidine (anti-His).