Figure 7.
Figure 7. PU.1 binds to MafB and inhibits its activity. (A) Analysis of PU.1 interaction with MafB in GST pull-down assays. In vitro–translated 35S-methionine-labeled PU.1 (lanes 1-3) or PUER (lanes 4-6) were incubated with an affinity matrix-bound GST-fusion protein of full-length MafB (lanes 3 and 6) or GST only control (lanes 2 and 5), washed, resuspended in sodium dodecyl sulfate (SDS) sample buffer, and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. (B) Effect of PU.1 on MafB transactivation activity. QT6 fibroblasts were cotransfected with 0.5 μg of a synthetic luciferase reporter construct containing 3 multimerized Maf response elements (3xMARE), 0.25 μg MafB expression construct, and 0.25 μg(+)or0.5 μg(++) PU.1 expression construct as indicated. (C) Effect of PU.1 on endogenous Maf transactivation activity in macrophages. HD11 macrophages (MΦ), expressing high levels of endogenous MafB39, were cotransfected with 0.5 μg of a synthetic luciferase reporter construct containing 3 multimerized Maf response elements (3xMARE), and 0.25 μg (+), 0.5 μg (++), or 1 μg (+++) PU.1 expression construct as indicated. Luciferase activities are expressed as fold activation over a reporter containing no response elements. Assays were performed in duplicate, normalized to β-galactosidase activity, and filled up with empty expression vector to a constant amount of expression plasmid. Error bars indicate standard error of the mean. Data are representative of at least 2 separate experiments. (D) PU.1 represses MafB-mediated macrophage differentiation. E26ts21-PUER clones were either treated (PU.1 active) or not (PU.1 inactive) with 1 μM βE for 48 hours, then shifted to 42°C or continued to be cultured at 37°C for an additional 48 hours. Expression of the macrophage-specific marker 47.83 was analyzed by FACS. (E) E26ts21-PUER clones were simultaneously treated with 1 μM βE and shifted to 42°C for 48 hours (42°C/βE) and compared to controls grown with βE or at 42°C only by FACS analysis for expression of 47.83. Results represent the average of at least 3 different clones. Error bars indicate standard error of the mean, and the statistical significance of the effect of 1 μM βE at 42°C is indicated.

PU.1 binds to MafB and inhibits its activity. (A) Analysis of PU.1 interaction with MafB in GST pull-down assays. In vitro–translated 35S-methionine-labeled PU.1 (lanes 1-3) or PUER (lanes 4-6) were incubated with an affinity matrix-bound GST-fusion protein of full-length MafB (lanes 3 and 6) or GST only control (lanes 2 and 5), washed, resuspended in sodium dodecyl sulfate (SDS) sample buffer, and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. (B) Effect of PU.1 on MafB transactivation activity. QT6 fibroblasts were cotransfected with 0.5 μg of a synthetic luciferase reporter construct containing 3 multimerized Maf response elements (3xMARE), 0.25 μg MafB expression construct, and 0.25 μg(+)or0.5 μg(++) PU.1 expression construct as indicated. (C) Effect of PU.1 on endogenous Maf transactivation activity in macrophages. HD11 macrophages (MΦ), expressing high levels of endogenous MafB39, were cotransfected with 0.5 μg of a synthetic luciferase reporter construct containing 3 multimerized Maf response elements (3xMARE), and 0.25 μg (+), 0.5 μg (++), or 1 μg (+++) PU.1 expression construct as indicated. Luciferase activities are expressed as fold activation over a reporter containing no response elements. Assays were performed in duplicate, normalized to β-galactosidase activity, and filled up with empty expression vector to a constant amount of expression plasmid. Error bars indicate standard error of the mean. Data are representative of at least 2 separate experiments. (D) PU.1 represses MafB-mediated macrophage differentiation. E26ts21-PUER clones were either treated (PU.1 active) or not (PU.1 inactive) with 1 μM βE for 48 hours, then shifted to 42°C or continued to be cultured at 37°C for an additional 48 hours. Expression of the macrophage-specific marker 47.83 was analyzed by FACS. (E) E26ts21-PUER clones were simultaneously treated with 1 μM βE and shifted to 42°C for 48 hours (42°C/βE) and compared to controls grown with βE or at 42°C only by FACS analysis for expression of 47.83. Results represent the average of at least 3 different clones. Error bars indicate standard error of the mean, and the statistical significance of the effect of 1 μM βE at 42°C is indicated.

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