Fig. 4: FOXQ1 binds to RbBP5 within the Forkhead box domain.

a Schematic of the FOXQ1 fragment subcloning. N-terminal (1-142), Forkhead box (105-227), and C-terminal (204-403) were fused to FLAG-tag. b Fragments were transfected into HEK293 cells with full-length FOXQ1 and vector control. Lysates were subject to IP with anti-FLAG resin and analyzed by western blot with an antibody against endogenous RbBP5. c Schematic of constructs designed to narrow the domain of interaction within FOXQ1., Additional constructs of the FOXQ1 N-terminal fragments (1–142) and the Forkhead box fragment (105-227) were transfected alongside C-terminal residues 142–403 and N-terminal 75–142 to refine the region responsible for the RbBP5 interaction. d IP western blot analysis with membranes probed with the RbBP5 antibody. *Indicates predicted band. e Analysis of the evolutionary conservation of the H1-S1 region of the FOXQ1 Forkhead box. Red cylinders indicate α-helix domains, blue rectangles indicate β-sheets, and purple triangles indicate winged regions. The highlighted residues alter RbBP5 binding upon mutation. f Schematic of site-directed mutagenesis within the FOXQ1 Forkhead box. g WT and mutant FOXQ1 vectors were transfected into HEK293 cells. Lysates were subject to anti-V5 IP, and the effects of the point mutation of FOXQ1 on RbBP5 binding were analyzed by western blot. For panels b, d, g, the image serves as a representative result from experiments performed in triplicate (n = 3). Unprocessed western blot images are provided in Source data 2.