Fig. 4. The nht and rye proteins physically interact with each other but not with their generally expressed binding partner homologs TAF12 and TAF4 in a bacterial co-expression and GST pulldown assay. Western blots of total bacterial protein extracts (T) and elutions of glutathione-Sepharose purified protein (B) from strains expressing GST or FLAG-tagged fusion proteins. Blots were probed sequentially with anti-GST or anti-FLAG antibodies. Only proteins that were soluble in the bacterial extracts were able to be assayed for binding to glutathione-Sepharose. Lanes: 1, GST-Nht; 2, FLAG-Rye; 3, GST-Nht and FLAG-Rye; 4, GST-dTAF4 and FLAG-Rye; 5, GST-dTAF4; 6, FLAG-dTAF12; 7, GST-dTAF4 and FLAG-dTAF12; 8, GST-Nht and FLAG-dTAF12. In all cases, bacterial strains expressing a single fusion protein also carried the second vector without a fusion protein insert.