Figure 2
Figure 2. C/EBPα-Cm or C/EBPα-Nm inhibited the transcriptional activation of C/EBPα-WT by different mechanisms. (A-B top) 293T cells were transiently transfected with indicated amounts of expression plasmids (pMXs-Flag-tagged C/EBPα WT-IP, pMXs-Myc-tagged C/EBPα mutants-IP, pMXs-IP, pEF-BOS/PU.1, pEF-BOS) together with 100 ng of the luciferase reporter plasmid p(C/EBP)2TK. The total amount of plasmid for each transfection was adjusted by adding empty plasmids (pMXs-IP or pEF-BOS). Results represented the average values for relative luciferase activity that were normalized using the activity of EF1 vector as an internal control. All transfection groups were normalized with a Renilla luciferase vector as an internal control. All data points correspond to the mean and the standard deviation (SD). Data are representative of 3 independent experiments. Statistically significant differences are shown. *P < .05. (Bottom) Expression of C/EBPα-WT, C/EBPα-mutants, or PU.1 in 293T cells transiently transfected as above described. Cell lysates were subject to immunoblotting with anti-Flag Ab, anti-Myc Ab, anti-PU.1 Ab, or anti-tubulin Ab as control. The results shown are representative of 3 independent experiments. (C) DNA binding of C/EBPα-WT and mutants. Electrophoresis mobility shift assay was performed with 32P-labeled oligonucleotides containing the C/EBPα binding site derived from CSF3R promoter and nuclear extracts from 293T cells transiently transfected with pMXs-Flag–tagged C/EBPα WT-IP, pMXs-Myc–tagged C/EBPα-Cm-IP, or pMXs-Myc–tagged C/EBPα-Nm-IP. Data are representative of 3 independent experiments. Lane 1: none (−); lane 2: control rabbit immunoglobulin G was added; lane 3: anti-C/EBPα Ab was added; lane 4: cold competitor (C.C) was added. Ss indicates supershifted bands. (D) 293T cells transiently transfected with pMXs-Flag–tagged C/EBPα-WT-IP, pMXs-Myc-tagged C/EBPα-Cm-IP, or pMXs-Myc–tagged C/EBPα-p30-IP were immunostained with anti-Flag Ab (red) or anti–c-Myc Ab (green) and stained with Hoechst (H33342; blue). Data are representative of 4 independent experiments (total of 15 mitotic cells were examined for each transfectant). Fluorescence images by confocal microscopy were obtained with IX70 (Olympus). Original magnification ×60.

C/EBPα-Cm or C/EBPα-Nm inhibited the transcriptional activation of C/EBPα-WT by different mechanisms. (A-B top) 293T cells were transiently transfected with indicated amounts of expression plasmids (pMXs-Flag-tagged C/EBPα WT-IP, pMXs-Myc-tagged C/EBPα mutants-IP, pMXs-IP, pEF-BOS/PU.1, pEF-BOS) together with 100 ng of the luciferase reporter plasmid p(C/EBP)2TK. The total amount of plasmid for each transfection was adjusted by adding empty plasmids (pMXs-IP or pEF-BOS). Results represented the average values for relative luciferase activity that were normalized using the activity of EF1 vector as an internal control. All transfection groups were normalized with a Renilla luciferase vector as an internal control. All data points correspond to the mean and the standard deviation (SD). Data are representative of 3 independent experiments. Statistically significant differences are shown. *P < .05. (Bottom) Expression of C/EBPα-WT, C/EBPα-mutants, or PU.1 in 293T cells transiently transfected as above described. Cell lysates were subject to immunoblotting with anti-Flag Ab, anti-Myc Ab, anti-PU.1 Ab, or anti-tubulin Ab as control. The results shown are representative of 3 independent experiments. (C) DNA binding of C/EBPα-WT and mutants. Electrophoresis mobility shift assay was performed with 32P-labeled oligonucleotides containing the C/EBPα binding site derived from CSF3R promoter and nuclear extracts from 293T cells transiently transfected with pMXs-Flag–tagged C/EBPα WT-IP, pMXs-Myc–tagged C/EBPα-Cm-IP, or pMXs-Myc–tagged C/EBPα-Nm-IP. Data are representative of 3 independent experiments. Lane 1: none (−); lane 2: control rabbit immunoglobulin G was added; lane 3: anti-C/EBPα Ab was added; lane 4: cold competitor (C.C) was added. Ss indicates supershifted bands. (D) 293T cells transiently transfected with pMXs-Flag–tagged C/EBPα-WT-IP, pMXs-Myc-tagged C/EBPα-Cm-IP, or pMXs-Myc–tagged C/EBPα-p30-IP were immunostained with anti-Flag Ab (red) or anti–c-Myc Ab (green) and stained with Hoechst (H33342; blue). Data are representative of 4 independent experiments (total of 15 mitotic cells were examined for each transfectant). Fluorescence images by confocal microscopy were obtained with IX70 (Olympus). Original magnification ×60.

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