The acidic tail (AT, residues 371-385), and c-terminal (Pelo-1, residues 268-371) region of PELO is required for binding to HAX1, and EIF3G and SPRX, respectively. A, To determine the regions of PELO that are required for its association with HAX1, EIF3G and SRPX, four GST-PELO fusion proteins were generated (GST-1, GST-2, GST-3 and GST-4) and tested for their ability to bind in GST pull-down assays. GST alone (GST-0) was used as control in the assay. B, Recombinant GST-PELO fusion proteins were subjected to SDS-PAGE and stained with Coomassie blue. C, Equivalent amounts of GST-PELO and GST-0 proteins bound to glutathione-matrices were subsequently incubated with extracts of HeLa cells, which were transfected with either Myc-tagged HAX1, EIF3G or SRPX constructs. Western blots containing the GST pull-down assays were incubated with monoclonal anti-Myc antibodies. Immunoblot analysis revealed that GST-1 and GST-2, but not GST-3, GST-4 and GST-0 bound to Myc-HAX1 protein. Except GST-4 and GST-0, all other GST-PELO fusion proteins exhibit specific association to Myc-EIF3G and Myc-SRPX.
Burnicka-Turek et al. BMC Cell Biology 2010 11:28 doi:10.1186/1471-2121-11-28