Journal: International immunology

Article Title: Inducible expression of nuclear factor IL-6 increases endogenous gene expression of macrophage inflammatory protein-1 alpha, osteopontin and CD14 in a monocytic leukemia cell line.

doi: 10.1093/intimm/10.12.1825

Figure Lengend Snippet: Fig. 1. Induction of lysozyme mRNA by stimulating NF-IL6 expression in M1 cells. (A) M1 cells stably transfected with an IPTG-inducible pOPRSVI-NFIL6 plasmid (NFIL6 M1 cells) were grown at 1.03105/ml and then 1 mM IPTG was added to the medium to stimulate NF-IL6 expression. Cell lysates collected at the indicated time points were tested by immunoblotting with anti-C/EBPβ polyclonal antibody. (B) Total RNA (15 µg/lane) was extracted from NFIL6 M1 cells at the indicated time points following stimulation with 1 mM IPTG and subjected to Northern blot hybridization with mouse lysozyme probe. The bottom panel shows a control hybridization with EF-1α-specific probe.

Article Snippet: After boiling for 5 min, cell lysates (13105 cells per lane) were separated by SDS–PAGE (4–20% gradient polyacrylamide gel), electrophoretically transferred to a nitrocellulose membrane and then analyzed for immunoreactivity with anti-C/EBPβ (NF-IL6) antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) by the enhanced chemiluminescence detection system (Dupont, Boston, MA).

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Northern Blot, Hybridization, Control

Journal: International immunology

Article Title: Inducible expression of nuclear factor IL-6 increases endogenous gene expression of macrophage inflammatory protein-1 alpha, osteopontin and CD14 in a monocytic leukemia cell line.

doi: 10.1093/intimm/10.12.1825

Figure Lengend Snippet: Fig. 3. NF-IL6 stimulates transcription of the MIP-1α promoter in transactivation assays. Murine MIP-1α reporter gene constructs (from top: MIP-1α promoter construct p-676Luc, p-341Luc, p-172Luc, p-99Luc, promoterless luciferase construct pGL3Basic) are shown schematically on the left. Numbers indicate positions relative to the transcription start site. The results of luciferase assays are illustrated on the right. NFIL6 M1 cells were co-transfected with 20 µg of these constructs and 2 µg of a sea-pansy luciferase vector (pRL-TK) as a transfection efficiency control. The cells were split into two equal parts post-transfection and cultured in the presence (1 mM) or absence of IPTG. The cultures were harvested 20 h after transfection and analyzed for luciferase activity. The promoter activity was determined as relative light units compared with the promoterless pGL3 basic vector. The data represent the mean 6 SD of three independent experiments.

Article Snippet: After boiling for 5 min, cell lysates (13105 cells per lane) were separated by SDS–PAGE (4–20% gradient polyacrylamide gel), electrophoretically transferred to a nitrocellulose membrane and then analyzed for immunoreactivity with anti-C/EBPβ (NF-IL6) antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) by the enhanced chemiluminescence detection system (Dupont, Boston, MA).

Techniques: Construct, Luciferase, Transfection, Plasmid Preparation, Control, Cell Culture, Activity Assay

Journal: International immunology

Article Title: Inducible expression of nuclear factor IL-6 increases endogenous gene expression of macrophage inflammatory protein-1 alpha, osteopontin and CD14 in a monocytic leukemia cell line.

doi: 10.1093/intimm/10.12.1825

Figure Lengend Snippet: Fig. 4. EMSA of the putative C/EBP binding sequences within the murine MIP-1α proximal promoter. (A) Nucleotide sequences of the murine MIP-1α proximal promoter. The transcriptional start site is overlined by a forward arrow and designated as position 11. The dashed underlines indicate putative C/EBP binding sites described previously (38). Sequences used to generate double-stranded oligonucleotides for EMSA studies are indicated by lines above the sequences, and designated A, B, C and D. Each oligonucleotide covers one C/EBP binding site. (B) The double-stranded A–D oligonucleotides were 32P-labeled and incubated with nuclear protein prepared from control NFIL6 M1 cells (lanes 1, 5, 9 and 13) or 12 h IPTG-treated NFIL6 M1 cells (lanes 2–4, 6–8, 10–12 and 14–16). The double-stranded competitor oligonucleotide from the human IL-6 promoter, which contains one NF-IL6 binding site, was added at a 50-fold molar excess over probe oligonucleotides (lanes 3, 7, 11 and 15). In lanes 4, 8, 12 and 16, rabbit polyclonal antibody to NF-IL6 was added to the binding reaction. The positions of free probe (F) and supershifted complex (S) are marked on the right side of each panel. The positions of DNA–nuclear protein complexes detected with probe A, B and D are also indicated on the right sides.

Article Snippet: After boiling for 5 min, cell lysates (13105 cells per lane) were separated by SDS–PAGE (4–20% gradient polyacrylamide gel), electrophoretically transferred to a nitrocellulose membrane and then analyzed for immunoreactivity with anti-C/EBPβ (NF-IL6) antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) by the enhanced chemiluminescence detection system (Dupont, Boston, MA).

Techniques: Binding Assay, Labeling, Incubation, Control

Journal: International immunology

Article Title: Inducible expression of nuclear factor IL-6 increases endogenous gene expression of macrophage inflammatory protein-1 alpha, osteopontin and CD14 in a monocytic leukemia cell line.

doi: 10.1093/intimm/10.12.1825

Figure Lengend Snippet: Fig. 5. Transactivation of MIP-1α promoter by NF-IL6 requires two NF-IL6 binding sites. (A) Competition analysis of the mutated NF-IL6 binding sites using an EMSA. The double-stranded B and D oligonucleotides were 32P labeled and incubated with nuclear protein prepared from control NFIL6 M1 cells (lanes 1 and 5) or 12 h IPTG-treated NFIL6 M1 cells (lanes 2–4 and 6–8). A 50-fold molar excess of unlabeled wild- type oligonucleotides B or D (lanes 3 and 7) and NF-IL6 binding site mutant oligonucleotides mB or mD (lanes 4 and 8) were added to the binding reaction. Symbols marked on the right side of each panel are as described in the legend to Fig. 4. (B) Murine MIP-1α reporter gene constructs (from top: MIP-1α promoter construct p-172Luc, position –115 to –114 NF-IL6 binding site mutated construct pMutBLuc and position –54 to –53 NF-IL6 binding site mutated construct pMutDLuc, promoterless luciferase construct pGL3Basic) are shown schematically on the left. Numbers indicate positions relative to the transcription start site. The closed circles B and D represent NF-IL6 binding sites located within oligonucleotides B and D used in EMSA respectively. The results of luciferase assays are illustrated on the right. NFIL6 M1 cells were co-transfected with 20 µg of these constructs and 2 µg of a sea-pansy luciferase vector (pRL-TK) as a transfection efficiency control. The cells were split into two equal parts post-transfection and cultured in the presence (1 mM) or absence of IPTG. The cultures were harvested 20 h after transfection and analyzed for luciferase activity. The promoter activity was determined as relative light units compared with the promoterless pGL3 basic vector. The data represent the mean 6 SD of three independent experiments.

Article Snippet: After boiling for 5 min, cell lysates (13105 cells per lane) were separated by SDS–PAGE (4–20% gradient polyacrylamide gel), electrophoretically transferred to a nitrocellulose membrane and then analyzed for immunoreactivity with anti-C/EBPβ (NF-IL6) antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) by the enhanced chemiluminescence detection system (Dupont, Boston, MA).

Techniques: Binding Assay, Labeling, Incubation, Control, Mutagenesis, Construct, Luciferase, Transfection, Plasmid Preparation, Cell Culture, Activity Assay

Journal: Nucleic Acids Research

Article Title: Shwachman–Bodian–Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs

doi: 10.1093/nar/gkw005

Figure Lengend Snippet: Deregulated C/EBPβ protein isoform expression in SDS. ( A ) The human C/EBPα and - β mRNAs are presented with consecutive translation initiation sites (arrowheads) and each of the protein isoforms and its size (*size of murine orthologs). Extended, p42, LAP* or LAP proteins are expressed through regular translation initiation, omitting the uORF. Truncated p30 or LIP proteins are expressed through translation re-initiation by post-translation ribosomes that first have translated the uORF. For detailed description of the uORFs and surrounding sequences, see ( – ). Expression of the Extended-C/EBPα isoform is generally weak because it uses the alternative GUG (CUG for murine) codon. Similarly, expression of the C/EBPβ-LAP* from a non-Kozak AUG codon is mostly weak. ( B ) SBDS protein levels were detected in SDS patient-derived (SW18, SW74) and healthy control-derived (wt) lymphoblastoid cells by immunoblotting. Long exposure shows the very low expression of wt SBDS in SW74 cells harbouring the homozygous 258 + 2T > C mutation. ( C ) The upper panels show immunoblots of C/EBPβ isoforms, SBDS and α-tubulin as loading control in both SDS patient-derived cells (SW18, SW74) and healthy control-derived cells (wt). The lower panels show immunoblots of 4E-BP1, phosphorylated-4EBP1 (P-4E-BP1), S6K1, phosphorylated-S6K1 (P-S6K1) and β-actin as loading control to monitor alterations in mTORC1 signalling ( D ) qRT-PCR analysis for endogenous C/EBPβ mRNA levels in patient-derived cells (SW18, SW74) and healthy control cells (wt). ( E ) Immunoblots for MYC, SBDS and α-tubulin (loading control) in SDS patient-derived and healthy control-derived cells. ( F ) qRT-PCR analysis of MYC transcript levels in SDS patient-derived and healthy control-derived cells. ( G ) Cell multiplication assay with patient-derived cells (SW18, SW74) and healthy control-derived cells (wt). Seven days after seeding 5 × 10 5 cells, cells were harvested and counted. Statistical differences were determined by Student's t -tests. Error bars represent ±SD (n = 3), * P < 0.05, ** P < 0.01.

Article Snippet: The primary antibodies, SBDS (S-15), C/EBPα C18 (sc-9314) for human C/EBPα and C/EBPα 14AA (SC-61) for rat C/EBPα, C/EBPβ C19 (SC-150) for both human and rat C/EBPβ, MYC (sc-40), α-Tubulin (sc-8035), 4E-BP1 (C-19) (all Santa Cruz Biotechnology) and Phosphorylated 4E-BP1 (Thr37/46) (9459), Phosphorylated p70-S6K (Thr389) (9234), p70-S6K (9202) (Cell Signaling) and β-actin (691001) (MP Biomedicals) were diluted in blocking buffer (1:400–1:10000).

Techniques: Expressing, Derivative Assay, Control, Western Blot, Mutagenesis, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Shwachman–Bodian–Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs

doi: 10.1093/nar/gkw005

Figure Lengend Snippet: SBDS is required for efficient C/EBPα-p30 and C/EBPβ-LIP expression. Expression of the endogenous ( A ) human C/EBPα (p42 and p30) or ( B ) human C/EBPβ proteins (43 kDa LAP and 20 kDa LIP) in HL-60 cells with stable SBDS knockdown (shSBDS) or control (scrambled shRNA) by immunoblotting, and quantification of p30/p42 or LIP/LAP ratio and qRT-PCR analysis of transcript levels at the right. The lower panels in (A) show immunoblots of 4E-BP1, phosphorylated-4E-BP1 (P-4E-BP1), S6K1, phosphorylated-S6K1 (P-S6K1) and β-actin as loading control to monitor alterations in mTORC1 signalling. Immunoblots of ( C ) rat C/EBPα (p42 and p30) or ( D ) rat C/EBPβ (38 kDA LAP*, 34 kDa LAP and 20 kDa LIP) expression in C33A cells with stable SBDS knockdown (shSBDS) and control (scrambled shRNA) transiently transfected with wild-type rat C/ebpα or wild-type rat C/ebpβ expression plasmids, respectively. Quantification of isoform ratio is shown at the top. The lower panels show immunoblots of 4E-BP1, phosphorylated-4E-BP1 (P-4E-BP1), S6K1, phosphorylated-S6K1 (P-S6K1) and β-actin as loading control to monitor alterations in mTORC1 signalling. ( E ) Immunoblot of rat C/EBPα or ( F ) rat C/EBPβ proteins in C33A cells transiently transfected with mutated uORF-AUG (dysfunctional uORF) rat C/ebpα or - β cDNA expression vectors. Statistical differences in the mRNA levels and isoform ratios were determined by Student's t -tests. Error bars represent ±SD (n = 3), * P < 0.05, ** P < 0.01, *** P < 0.005.

Article Snippet: The primary antibodies, SBDS (S-15), C/EBPα C18 (sc-9314) for human C/EBPα and C/EBPα 14AA (SC-61) for rat C/EBPα, C/EBPβ C19 (SC-150) for both human and rat C/EBPβ, MYC (sc-40), α-Tubulin (sc-8035), 4E-BP1 (C-19) (all Santa Cruz Biotechnology) and Phosphorylated 4E-BP1 (Thr37/46) (9459), Phosphorylated p70-S6K (Thr389) (9234), p70-S6K (9202) (Cell Signaling) and β-actin (691001) (MP Biomedicals) were diluted in blocking buffer (1:400–1:10000).

Techniques: Expressing, Knockdown, Control, shRNA, Western Blot, Quantitative RT-PCR, Transfection

Journal: Nucleic Acids Research

Article Title: Shwachman–Bodian–Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs

doi: 10.1093/nar/gkw005

Figure Lengend Snippet: SBDS-dependent C/EBPα p30/p42 isoform ratio regulates MYC expression and cell proliferation in HL-60 cells. ( A ) Immunoblots showing endogenous MYC, C/EBPα, SBDS and α-Tubulin (loading control) protein expression in HL-60 cells with stable knockdown of SBDS (shSBDS) or control (scrambled shRNA). Quantification of the p30/p42 isoform ratio and MYC protein levels are shown on the right panel. ( B ) qRT-PCR analysis of endogenous MYC mRNA expression level in HL-60 cells and HL-60-p30flag overexpressing cells with stable knockdown of SBDS (shSBDS) or control (scrambled shRNA). ( C ) HL-60 cells stably overexpressing C/EBPα-p30flag and knockdown of SBDS (shSBDS) or control (scrambled shRNA). Immunoblot shows endogenous MYC, C/EBPα, SBDS and α-tubulin (loading control) protein expression. Quantification of the p30/p42 isoform ratio and MYC protein levels are shown on the right panel. ( D ) Cell multiplication assay with HL-60 cells expressing scrambled shRNA (control), shSBDS ( SBDS knockdown), shSBDS/C/EBPα-p30flag or scrambled shRNA/C/EBPα-p30flag. Seven days after seeding 5 × 10 5 cells, cells were harvested and counted. Different letters (a, b, c, d) indicate significant differences in cell numbers determined by one-way-ANOVA. Error bars represent ±SD (n = 3), P < 0.05. ( E ) HL-60 cells were treated with 1 μM PP242 or DMSO as a control for 48 h. Immunoblots show expression of MYC, p42, p30flag, p30, p4E-BP1, 4E-BP1, SBDS and α-Tubulin (loading control). The phosphorylation levels of 4E-BP1 indicate mTORC1 activity. Quantification of the p30/p42 isoform ratio and MYC protein levels are shown on the right panel. ( F ) Endogenous MYC mRNA levels by qRT-PCR. ( G ) Cell multiplication assay with HL-60 cells incubated with 1 μM PP242 or DMSO (solvent). Seven days after seeding 5 × 10 5 cells, cells were harvested and counted. ( H ) HL-60-p30flag overexpressing cells were treated with 1 μM PP242 or DMSO for 48 h and expression of MYC, p42, p30flag, p30, p4E-BP1, 4E-BP1, SBDS and α-Tubulin (loading control) was examined by immunoblotting. Quantification of the p30/p42 isoform ratio and MYC protein levels are shown on the upper panel. ( I ) Endogenous MYC mRNA transcription levels and ( J ) the proliferation capacity were measured as described above. For all panels except for panel D, statistical differences were determined by Student's t -tests. Error bars represent ±SD (n = 3), * P < 0.05, ** P < 0.01, *** P < 0.005.

Article Snippet: The primary antibodies, SBDS (S-15), C/EBPα C18 (sc-9314) for human C/EBPα and C/EBPα 14AA (SC-61) for rat C/EBPα, C/EBPβ C19 (SC-150) for both human and rat C/EBPβ, MYC (sc-40), α-Tubulin (sc-8035), 4E-BP1 (C-19) (all Santa Cruz Biotechnology) and Phosphorylated 4E-BP1 (Thr37/46) (9459), Phosphorylated p70-S6K (Thr389) (9234), p70-S6K (9202) (Cell Signaling) and β-actin (691001) (MP Biomedicals) were diluted in blocking buffer (1:400–1:10000).

Techniques: Expressing, Western Blot, Control, Knockdown, shRNA, Quantitative RT-PCR, Stable Transfection, Phospho-proteomics, Activity Assay, Incubation, Solvent

Journal: BMC Molecular Biology

Article Title: TGFβ1 enhances MAD1 expression and stimulates promoter-bound Pol II phosphorylation: basic functions of C/EBP, SP and SMAD3 transcription factors

doi: 10.1186/1471-2199-12-9

Figure Lengend Snippet: Induction of MAD1 mRNA and protein expression in response to TGFβ1 in U937 cells . A . Exponentially proliferating U937 promyelocytes were treated with 2.5 ng/ml of TGFβ1. mRNA was purified at the indicated time points. The kinetics of MAD1 expression in response to TGFβ1 were analyzed by quantitative RT-PCR. The mean values ± SD of three independent experiments are shown. B . Whole cell extracts of U937 cells treated as in panel A were generated at the indicated time points and analyzed for endogenous MAD1 protein by western blotting. Tubulin served as loading control. C . U937 cells were pre-treated with the TGFβ receptor I inhibitor SB505124 (5 μM) for 40 min prior to addition of TGFβ1. MAD1 expression was assayed by quantitative RT-PCR 2 hrs after TGFβ1 stimulation. The displayed data are mean values ± SD of three independently performed experiments. D . U937 cells were initially diluted to a density of 10 5 cells/ml and incubated with 2.5 ng/ml TGFβ1 over a period of 48 hrs. The number of cells was determined at the indicated time-points by CASY measurements.

Article Snippet: Whole cell extracts were resolved by SDS-Page and transferred onto nitrocellulose membranes, probed with MAD1 C19 (SC-222, Santa Cruz Biotechnology), α-Tubulin (B-5-1-2, T-5168, Sigma), or C/EBPβ C19 (SC-150, Santa Cruz Biotechnology) antibodies followed by horseradish peroxidase (HRP)-labeled secondary antibody.

Techniques: Expressing, Purification, Quantitative RT-PCR, Generated, Western Blot, Control, Incubation

Journal: BMC Molecular Biology

Article Title: TGFβ1 enhances MAD1 expression and stimulates promoter-bound Pol II phosphorylation: basic functions of C/EBP, SP and SMAD3 transcription factors

doi: 10.1186/1471-2199-12-9

Figure Lengend Snippet: MAD1 promoter reporter gene constructs are regulated by C/EBP transcription factors . A . Schematic representation of the position of the CCAAT box (half-sites) and GC box motifs within the MAD1 promoter region. The major transcriptional start site is indicated with +1. B . and C . HeLa cells were co-transfected with the -1284 to +248 or the -184 to +248 reporter gene constructs, respectively, and plasmids encoding the three indicated C/EBP proteins. The promoter-less pGL2 construct served as negative control and its activity was set as 1. The luciferase measurements were normalized to β-galactosidase activities expressed from a co-transfected plasmid. D . Reporter gene assays were performed in HeLa cells by co-transfecting the -184 to +248 reporter gene construct and a pSuper-based shRNA targeting C/EBPβ or a control shRNA. Luciferase activity was determined 2 days after transfection. E . The efficiency of the shRNA targeting C/EBPβ was examined in HeLa cells by immunoblotting. C/EBPβ-specific antibodies were used to detect the two major isoforms of endogenous C/EBPβ proteins, LAP and LIP, in whole cell extracts. F . The C/EBPβ-mediated relative activation of the indicated MAD1 promoter constructs were analyzed in reporter gene assays performed in HeLa cells as described above.

Article Snippet: Whole cell extracts were resolved by SDS-Page and transferred onto nitrocellulose membranes, probed with MAD1 C19 (SC-222, Santa Cruz Biotechnology), α-Tubulin (B-5-1-2, T-5168, Sigma), or C/EBPβ C19 (SC-150, Santa Cruz Biotechnology) antibodies followed by horseradish peroxidase (HRP)-labeled secondary antibody.

Techniques: Construct, Transfection, Negative Control, Activity Assay, Luciferase, Plasmid Preparation, shRNA, Control, Western Blot, Activation Assay

Journal: BMC Molecular Biology

Article Title: TGFβ1 enhances MAD1 expression and stimulates promoter-bound Pol II phosphorylation: basic functions of C/EBP, SP and SMAD3 transcription factors

doi: 10.1186/1471-2199-12-9

Figure Lengend Snippet: C/EBPβ binds to the chromatin embedded MAD1 promoter in cells . A . Schematic presentation of the MAD1 promoter relative to the major transcriptional start site with the homology region as defined in . The positions of primer sets used for ChIP PCR are given (upper panel). U937 cells were used in ChIP assays to investigate the binding of C/EBPβ to the MAD1 promoter in response to TGFβ1. The cells were treated with 2.5 ng/ml TGFβ1 for 1.5 hrs prior to crosslinking and immunoprecipitation, and the purified DNA was analyzed by PCR with the indicated primers. The displayed experiment is representative of three independent experiments with similar outcome. B . C/EBPβ was expressed in HEK293 cells. Whole cell extracts were generated and incubated with [ 32 P]-radiolabelled CCAAT box1 oligonucleotids derived from the MAD1 or the neutrophil elastase promoter (NE-CCAAT). Supershift experiments with C/EBPβ specific antibodies are indicated. The complexes were analyzed by EMSA and detected by autoradiography. Ø, indicates lanes without cell extracts; +, whole cell extracts of control transfected HEK293 cells; β, whole cell extracts of HEK293 cells transiently expressing C/EBPβ. C . The C/EBPα/β heterodimer DNA-binding activity to CCAAT box1 was determined by EMSA. HEK293 cell extracts overexpressing C/EBPα and C/EBPβ were generated as in panel B and incubated with radiolabelled CCAAT box1. Supershift experiments were performed with C/EBPα- or C/EBPβ-specific antibody. For control an irrelevant oligonucleotide was used. D . Re-ChIP experiment using C/EBPα- and C/EBPβ-specific antibodies in U937 cells treated with or without TGFβ1 for 1.5 hrs. The first immunoprecipitation was performed with antibodies specific for C/EBPβ. The protein-DNA complexes were then released and subjected to a second immunoprecipitation using C/EBPα or C/EBPβ specific antibodies. The isolated DNA was analyzed by quantitative PCR with the primer set P (panel A) amplifying a portion of the homology region of the MAD1 promoter. The signal intensities obtained with the specific immunoprecipitations were normalized to samples generated with control antibodies. For all panels one representative of typically at least three independent experiments are shown.

Article Snippet: Whole cell extracts were resolved by SDS-Page and transferred onto nitrocellulose membranes, probed with MAD1 C19 (SC-222, Santa Cruz Biotechnology), α-Tubulin (B-5-1-2, T-5168, Sigma), or C/EBPβ C19 (SC-150, Santa Cruz Biotechnology) antibodies followed by horseradish peroxidase (HRP)-labeled secondary antibody.

Techniques: Binding Assay, Immunoprecipitation, Purification, Generated, Incubation, Derivative Assay, Autoradiography, Control, Transfection, Expressing, Activity Assay, Isolation, Real-time Polymerase Chain Reaction

Journal: BMC Molecular Biology

Article Title: TGFβ1 enhances MAD1 expression and stimulates promoter-bound Pol II phosphorylation: basic functions of C/EBP, SP and SMAD3 transcription factors

doi: 10.1186/1471-2199-12-9

Figure Lengend Snippet: Constitutive SP1 and SP3 binding to the chromatin embedded MAD1 promoter . A . Nuclear extracts of U937 cells were incubated with radiolabelled GC box1 and supershift experiments were performed with SP1 (α-SP1-1, SC-59; α-SP1-2, obtained from G. Suske) and SP3 specific antibodies. The EMSA was performed as described in the legend to Figure 3. B . Schematic representation of the positions of primer sets used for ChIP PCR relative to the major transcriptional start site within the MAD1 gene (upper panel). U937 cells were treated with 2.5 ng/ml TGFβ1 for 1.5 h, cross-linked and examined for SP1 and SP3 binding by ChIP. The isolated DNA was analyzed by PCR using the indicated primer sets.

Article Snippet: Whole cell extracts were resolved by SDS-Page and transferred onto nitrocellulose membranes, probed with MAD1 C19 (SC-222, Santa Cruz Biotechnology), α-Tubulin (B-5-1-2, T-5168, Sigma), or C/EBPβ C19 (SC-150, Santa Cruz Biotechnology) antibodies followed by horseradish peroxidase (HRP)-labeled secondary antibody.

Techniques: Binding Assay, Incubation, Isolation

Journal: BMC Molecular Biology

Article Title: TGFβ1 enhances MAD1 expression and stimulates promoter-bound Pol II phosphorylation: basic functions of C/EBP, SP and SMAD3 transcription factors

doi: 10.1186/1471-2199-12-9

Figure Lengend Snippet: SP1 and C/EBPβ cooperate in MAD1 promoter reporter gene activation . A . HeLa cells were co-transfected with the -184 to +248 reporter gene construct and C/EBPβ and/or SP1 expression vectors as indicated. B . A dominant negative SP1 mutant (SP1dn) was co-expressed with C/EBPβ in HeLa cells and the activity of the -184 to +248 reporter gene construct was determined. C . and D . HeLa cells were transiently transfected with the indicated expression plasmids and the -184 to +248 reporter gene construct or reporter constructs with the indicated mutations in the identified response elements.

Article Snippet: Whole cell extracts were resolved by SDS-Page and transferred onto nitrocellulose membranes, probed with MAD1 C19 (SC-222, Santa Cruz Biotechnology), α-Tubulin (B-5-1-2, T-5168, Sigma), or C/EBPβ C19 (SC-150, Santa Cruz Biotechnology) antibodies followed by horseradish peroxidase (HRP)-labeled secondary antibody.

Techniques: Activation Assay, Transfection, Construct, Expressing, Dominant Negative Mutation, Mutagenesis, Activity Assay

Journal: BMC Molecular Biology

Article Title: TGFβ1 enhances MAD1 expression and stimulates promoter-bound Pol II phosphorylation: basic functions of C/EBP, SP and SMAD3 transcription factors

doi: 10.1186/1471-2199-12-9

Figure Lengend Snippet: Influence of SMAD proteins and TGFβ1 receptor signaling on MAD1 promoter activity . A . HeLa cells were co-transfected with the -1282 to +248 reporter gene construct and SMADs and/or TGFβRca or TGFβR expression vectors as indicated. For control the pGL2 reporter was measured. B and C . As in A except that the SMAD binding element (SBE) reporter gene was used. D . The effect of TGFβRca and TGFβR was measured on different reporter gene constructs containing different fragments of the MAD1 promoter in HeLa cells (as indicated). E . As in panel D except that the effect of SMAD3 was determined on different fragments of the MAD1 promoter. F . The effect of SMAD3 was determined on the -184 to -58 fragment of the MAD1 promoter. This fragment contains the relevant C/EBP half sites and the GC box that interacts with SP factors. The fragments were fused to the minimal thymidine kinase promoter (minTK). ΔC/EBP1+2 refers to mutations of the two half sites that interact with C/EBP proteins and ΔGC-box to the mutation of the SP binding site as described preciously . G . HeLa cells were co-transfected with the -184 to +248 reporter gene construct and C/EBPα and SMAD3 as indicated. For control the pGL2 reporter was measured. H . U937 cells were treated for the indicated times with TGFβ1. The cells were then fixed in formaldehyde, lysed and the DNA fragmented. The binding of SMAD3 to the MAD1 promoter was measured after specific immunoprecipiation and analysis of the promoter region using the primers given in figure 4B (P and P'). For control an irrelevant promoter, MYOD1 , was used. The binding is given as % of input control.

Article Snippet: Whole cell extracts were resolved by SDS-Page and transferred onto nitrocellulose membranes, probed with MAD1 C19 (SC-222, Santa Cruz Biotechnology), α-Tubulin (B-5-1-2, T-5168, Sigma), or C/EBPβ C19 (SC-150, Santa Cruz Biotechnology) antibodies followed by horseradish peroxidase (HRP)-labeled secondary antibody.

Techniques: Activity Assay, Transfection, Construct, Expressing, Control, Binding Assay, Mutagenesis

Journal: BMC Molecular Biology

Article Title: TGFβ1 enhances MAD1 expression and stimulates promoter-bound Pol II phosphorylation: basic functions of C/EBP, SP and SMAD3 transcription factors

doi: 10.1186/1471-2199-12-9

Figure Lengend Snippet: Histone modifications and Pol II loading at the MAD1 promoter . A . Summary of primer sets used for ChIP PCR relative to the major transcriptional start site within the MAD1 gene. B . and C . Proliferating U937 cells were treated with or without 2.5 ng/ml TGFβ1 for 1.5 h. ChIP experiments were performed with the indicated antibodies and primers. For control the immunoprecipitations were performed with Protein A Sepharose beads only. Signals obtained from input DNA, given as part of the immunoprecipitated material, are shown for comparison. D . U937 cells were treated with or without 2.5 ng/ml TGFβ1 for 20 and 40 min prior to cross-linking. For ChIP, total Pol II and Ser-2 and Ser-5 phosphorylated Pol II was immunoprecipitated. Loading and distribution of the different Pol II isoforms were examined on the MAD1 gene with the indicated primers.

Article Snippet: Whole cell extracts were resolved by SDS-Page and transferred onto nitrocellulose membranes, probed with MAD1 C19 (SC-222, Santa Cruz Biotechnology), α-Tubulin (B-5-1-2, T-5168, Sigma), or C/EBPβ C19 (SC-150, Santa Cruz Biotechnology) antibodies followed by horseradish peroxidase (HRP)-labeled secondary antibody.

Techniques: Control, Immunoprecipitation, Comparison