Figure 3: The BAP1 and KLF5 proteins directly interact.

(a) Endogenous BAP1 and KLF5 proteins interact with one another in MCF10A cells. Endogenous BAP1 proteins were immunoprecipitated with the anti-BAP1 antibody. Immunoglobulin (Ig)G serves as the negative control. Endogenous KLF5 was detected by WB. (b) Purified recombinant GST-BAP interacts with KLF5-6 × His. GST-BAP1 and GST proteins were pulled down with glutathione beads. KLF5-6 × His was detected by WB. Purified GST and GST-BAP1 were detected by Coomassie blue staining. (c) Purified recombinant KLF5-6 × His interacts with GST-BAP. KLF5-6 × His proteins were pulled down with nickel beads. GST-BAP was detected by WB. (d) Mapping the domains of BAP1 that interact with KLF5. GST-fused full-length or deletion constructs of BAP1 (a schematic diagram is shown below the panel) were co-expressed with KLF5-3 × Flag in HEK293FT cells. KLF5-3 × Flag proteins were immunoprecipitated with Flag-M2 beads, and BAP1 proteins were detected by WB. (e) Mapping the domains of KLF5 that interact with BAP1. Flag-tagged full-length or deletion constructs of KLF5 (a schematic diagram is shown below the panel) and GST-BAP1 were transfected into HEK293FT cells. GST-BAP1 proteins were pulled down with glutathione beads, and KLF5 proteins were detected by WB. (f) BAP1 and KLF5 are co-localized in the nucleus of HCC1806 cells. The cellular location of KLF5 and BAP1 was examined by immunofluorescence staining. DAPI was used to stain the DNA. Scale bar, 10 μM. DAPI, 4,6-diamidino-2-phenylindole.