FIG. 2.

Effects of SET on KLF5 activity. (A) Effects of SET on KLF5 DNA-binding activity. A gel shift assay with recombinant KLF5 ZF/DBD and SET was performed. Wt and mut represent wild and mutant oligonucleotide competitors (lanes 5 and 6). The amount of recombinant protein is as follows: 150 (+) and 450 (++) ng for SET (lanes 2, 7, and 8) and 10 (+) and 50 (++) ng for KLF5 ZF/DBD (lanes 3 to 8). (B) Gel shift assay of control NF-κB p50 subunit and SET. Gel shift units (0.1) of NF-κB p50 (lanes 2, 4, and 5) and 100 (+) and 300 (++) ng of SET (lanes 3, 4, and 5) were used. (C) Effects of SET on KLF5 transactivation. Cotransfection analysis of effects of SET on KLF5 transcriptional activation.One hundred nanograms of reporter was used in each lane. Effectors are as follows: lanes 2, 3, and 4 through 7, and 12, 13, and 14 through 17 were 83, 250, and 750 ng of KLF5 expression plasmid (pCAG-KLF5), respectively; lanes 5 and 8, 6 and 9, 7 and 10, 15 and 18, 16 and 19, and 17 and 20 were 28, 83, and 250 ng of SET expression plasmid (pCHA-SET/TAF-Iβ). The total amount of effector plasmid was adjusted to 1 μg with the respective control vector. (D) Effects of SET on control NF-κB transactivation. NF-κB transactivation (lanes 2 to 5) was done by transfection of equal amounts (250 ng) of p50 and p65 subunit expression vectors. (E) Effects of SET on KLF5-induced cell growth. SET was transiently transfected into cells stably expressing epitope-tagged (3× FLAG) KLF5 or mock vector in 3T3-3 cells. The cell count on day 5 after transfection compared with mock vector-treated cells is shown. Error bars denote standard errors. (F) BrdU assay showing effects of KLF5 and SET on cell growth by use of adenovirus-mediated transfer (multiplicity of infection, 100) of KLF5 and SET adenoviruses. Empty (lane 1) denotes empty vector alone. Error bars denote standard errors. OD₄₅₀, optical density at 450 nm. (G) In vitro binding of KLF5 ZF/DBD and SET deletion mutants. Immobilized histidine-tagged SET protein was reacted with GST-KLF5 ZF/DBD fusion protein, separated by SDS-PAGE, and analyzed by immunoblotting with anti-GST antibody. SET deletion mutants are shown by their amino acid numbers in reference to the schematic diagram of functional domains shown above. Lane 1 is GST KLF5 ZF/DBD input, and lane 2 shows that GST KLF5 ZF/DBD does not bind Probond nickel-chelating resin. Lanes 3 to 8 show GST KLF5 ZF/DBD binding to respective resin-bound deletion mutants. Amino acids (aa) 1 to 24 comprise the SET-specific N-terminal region, amino acids 25 to 65 comprise the coiled-coil dimerization domain, amino acids 25 to 119 comprise a region known to inhibit phosphatase PP2A, amino acids 120 to 225 comprise a region with unknown function, and amino acids 226 to 277 comprise the acidic C-terminal region. All experiments were done at least twice with consistent findings.