Fig. 3. In vitro binding studies to validate the interacting interface residues predicted in the CTD/INI1_183-304 complex.

a Sequence of a portion of S6/Rpt1 fragment of INI1 and the IN-interaction-defective substitution mutations (E3, E4, and E10) identified in a random genetic, reverse yeast two-hybrid screen and their effect on S6-mediated inhibition of HIV-1 particle production. b GST-pull down assay to demonstrate the binding of INI1_183-304 and its mutants with IN, CCD and CTD. Representative images from one out of three experiment is shown. Top panel represents bound proteins and the bottom two panels represent the loading control. Top two panels represent the Western blot using α-BAF47 antibodies to detect 6His-SUMO-INI1_183-304. Bottom panel represents the Coomassie-stained gel of GST-fusion proteins. c GST-pull down assay to determine the interaction of His₆-IN(WT), His₆-IN(W235E) mutant with GST-INI1, GST-SAP18, GST-LEDGF, and GST-Gemin2. Representative images from one out of three experiment is shown. Top panel represents the bound proteins and the two panels below the top represent the loading controls. Non-adjacent lanes from the same gel are spliced together for the figure and uncropped gels are provided in the source data. Graphs at the bottom represent quantitation of the bound proteins expressed as fraction bound after normalizing to the loading control. The graphs represent the mean of two independent experiments, WT is Wild type IN (shown in blue) and W235E is shown in red.