Fig. 6.
Fig. 6. Direct physical interaction between KLF6 and Sp1. / (A-B) Coimmunoprecipitation experiments in mammalian cells. COS-7 cells were transfected with the KLF6 expression vectorpCIneo-KLF6, and 24 hours later their total lysates were immunoprecipitated (IP) with a rabbit polyclonal antibody against KLF6 (A) or against Sp1 (B). Specific immune complexes were isolated using protein G-Sepharose, washed, and electrophoresed in 8% SDS-PAGE gels. Proteins were blotted onto nitrocellulose, and the specific antigens were detected with the indicated rabbit polyclonal antibody, followed by a secondary goat-antirabbit coupled with horseradish peroxidase (HRP) and an enhanced chemiluminescence assay. Arrows indicate the bands of transfected KLF6, endogenous Sp1, and immunoglobulin chain coming from the antibody used in the immunoprecipitation (IgG). As a control for specificity, a parallel immunoprecipitation with a nonimmune rabbit immunoglobulin (IP IgG) was performed. WB indicates Western blotting. (C) Pull-down experiments. A mixture of Sp1 plus GST-KLF6 was incubated with either glutathione-Sepharose beads or anti-Sp1 antibody-conjugated agarose beads at 4°C overnight, as described in “Materials and methods.” Proteins were precipitated, and eluted proteins were analyzed by Western blotting with either anti-Sp1 or anti-KLF6 antibody. Lanes 1 to 4, probed with anti-Sp1 antibody; lanes 5 to 8, probed with anti-KLF6 antibody. Lanes 1 and 6, KLF6-GST; lanes 2 and 5, Sp1; lane 3, Sp1 preincubated together with KLF6 and precipitated with glutathione-Sepharose; lane 4, Sp1 incubated with glutathione-Sepharose without KLF6; lane 7, KLF6 preincubated together with Sp1 and precipitated with anti-Sp1 antibody; lane 8, KLF6 incubated with anti-Sp1 antibody without Sp1. The experiment was repeated 3 times with similar results, and representative results are shown. (D-E) Analysis of the interaction between Sp1 and KLF6 using the GAL4 one-hybrid system. (D) Sp1 and KLF6 functionally interact with each other when Sp1 is bound to DNA. HeLa cells were transfected with 0.5 μg GAL4-LUC reporter with or without 0.5 μg KLF6-GAL4, Sp1, KLF6, Sp1-GAL4, and Egr-1, as indicated. After 24 hours, luciferase activity was measured and normalized to the luciferase value obtained after transfection with GAL-4 LUC, which was given an arbitrary value of 1. (E) The C-terminal domain of Sp1 interacts with KLF6.Drosophila Schneider cells, SL-2, were transfected with 0.5 μg GAL4-LUC reporter with or without 0.5 μg GAL-4-KLF6 and 0.2 μg of full lengthpAC-Sp1 or deletion ΔΜ, ΔN, andΔC Sp1 mutants in the pAC vector. Luciferase activity was expressed as described for panel D.

Direct physical interaction between KLF6 and Sp1.

(A-B) Coimmunoprecipitation experiments in mammalian cells. COS-7 cells were transfected with the KLF6 expression vectorpCIneo-KLF6, and 24 hours later their total lysates were immunoprecipitated (IP) with a rabbit polyclonal antibody against KLF6 (A) or against Sp1 (B). Specific immune complexes were isolated using protein G-Sepharose, washed, and electrophoresed in 8% SDS-PAGE gels. Proteins were blotted onto nitrocellulose, and the specific antigens were detected with the indicated rabbit polyclonal antibody, followed by a secondary goat-antirabbit coupled with horseradish peroxidase (HRP) and an enhanced chemiluminescence assay. Arrows indicate the bands of transfected KLF6, endogenous Sp1, and immunoglobulin chain coming from the antibody used in the immunoprecipitation (IgG). As a control for specificity, a parallel immunoprecipitation with a nonimmune rabbit immunoglobulin (IP IgG) was performed. WB indicates Western blotting. (C) Pull-down experiments. A mixture of Sp1 plus GST-KLF6 was incubated with either glutathione-Sepharose beads or anti-Sp1 antibody-conjugated agarose beads at 4°C overnight, as described in “Materials and methods.” Proteins were precipitated, and eluted proteins were analyzed by Western blotting with either anti-Sp1 or anti-KLF6 antibody. Lanes 1 to 4, probed with anti-Sp1 antibody; lanes 5 to 8, probed with anti-KLF6 antibody. Lanes 1 and 6, KLF6-GST; lanes 2 and 5, Sp1; lane 3, Sp1 preincubated together with KLF6 and precipitated with glutathione-Sepharose; lane 4, Sp1 incubated with glutathione-Sepharose without KLF6; lane 7, KLF6 preincubated together with Sp1 and precipitated with anti-Sp1 antibody; lane 8, KLF6 incubated with anti-Sp1 antibody without Sp1. The experiment was repeated 3 times with similar results, and representative results are shown. (D-E) Analysis of the interaction between Sp1 and KLF6 using the GAL4 one-hybrid system. (D) Sp1 and KLF6 functionally interact with each other when Sp1 is bound to DNA. HeLa cells were transfected with 0.5 μg GAL4-LUC reporter with or without 0.5 μg KLF6-GAL4, Sp1, KLF6, Sp1-GAL4, and Egr-1, as indicated. After 24 hours, luciferase activity was measured and normalized to the luciferase value obtained after transfection with GAL-4 LUC, which was given an arbitrary value of 1. (E) The C-terminal domain of Sp1 interacts with KLF6.Drosophila Schneider cells, SL-2, were transfected with 0.5 μg GAL4-LUC reporter with or without 0.5 μg GAL-4-KLF6 and 0.2 μg of full lengthpAC-Sp1 or deletion ΔΜ, ΔN, andΔC Sp1 mutants in the pAC vector. Luciferase activity was expressed as described for panel D.

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