Protein-Protein Interaction Pulldown Assay

The assay was performed as follows^{25 (link)}. Briefly, the purified proteins were used to perform the pull-down assay in a reaction system comprising 800 µL PBS buffer, 5 µM (final concentration) MBP-RpfG and HtsH1C-Flag-His, HtsH2C-HA-His or HtsH3C-Myc-His proteins, and 50 µL Dextrin Sepharose High Performance (Sigma-Aldrich, St. Louis, MO, USA). All samples were incubated at 4 °C overnight. The agarose was collected by centrifugation and washed ten times with PBS containing 1% Triton X-100 to remove non-specifically bound proteins. The MBP-bead-captured proteins were eluted by boiling in 6× SDS loading dye for 10 min. These samples were subjected to SDS-PAGE and Western blotting. Protein detection involved the use of MBP-specific (ab49923), Flag-specific (ab1162), HA-specific (ab187915), Myc-specific (ab32072), and His-specific (ab18184) antibodies obtained from Abcam, UK.