Journal: Molecular and Cellular Biology

Article Title: Transforming Growth Factor ? Inhibits Bone Morphogenetic Protein-Induced Transcription through Novel Phosphorylated Smad1/5-Smad3 Complexes

doi: 10.1128/MCB.00231-12

Figure Lengend Snippet: TGF-β inhibits BMP-induced transcriptional responses in an ALK5-dependent manner. (A, B, and E to G) MDA-MB-231 BRE cells and MDA-MB-231 CAGA cells were induced with the ligands indicated and assayed for luciferase and Renilla activities after 8 h. (C) C2C12 and HaCaT cells were transiently transfected with BRE-luciferase and TK-Renilla. After 24 h, the C2C12 cells were induced with the ligands shown and luciferase/Renilla assays were performed. At 24 h after transfection, HaCaT cells were starved overnight in Opti-MEM before ligand induction. (D) MDA-MB-231 cells were transfected with ID2-luciferase and TK-Renilla for 24 h prior to ligand induction. In panel F, SB-431542 was added 15 min before the ligands shown, and in panel G, either dimethyl sulfoxide (DMSO) or SB-431542 was added 15, 30, or 60 min after ligand addition, as shown. All experiments were performed in triplicate. The data are presented as luciferase activity normalized to Renilla activity and are the mean and standard deviation from a single representative experiment. In panel E (right side), in parallel with the luciferase assay, MDA-MB-231 BRE cells were treated with ligands as indicated for 1 h. Whole-cell extracts were Western blotted using antibodies against PSmad1/5, PSmad2, and PSmad3, as well as total Smad1, -2, and -3. MCM7 is a loading control. The asterisk indicates a nonspecific band.

Article Snippet: The antibodies used were obtained from Santa Cruz Biotechnology (anti-Smad4 [B-8], anti-Smad3 [38-Q], anti-MCM7 [141.2], anti-MCM6 [C-20], and anti-Alk5 [V-22]), Sigma (anti-FLAG antibody [M2]), Life Technologies (anti-Smad1 [catalog no. 385400] and anti-Smad2 [catalog no. 511300]), Cell Signaling Technology (anti-Smad5 [catalog no. 9517], anti-phospho-Smad2 [catalog no. 3108], anti-phospho-Smad3 [catalog no. 9520S], and anti-phospho-Smad1/5/8 [catalog no. 9516]), Abcam (chromatin immunoprecipitation [ChIP] grade anti-Smad3 [ab28379] for immunoprecipitation [IP] and ChIP), Covance (RNA polymerase II [PolII; 8WG16]), and BD Biosciences (Smad2/3 [catalog no. 610843]).

Techniques: Luciferase, Transfection, Activity Assay, Standard Deviation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Transforming Growth Factor ? Inhibits Bone Morphogenetic Protein-Induced Transcription through Novel Phosphorylated Smad1/5-Smad3 Complexes

doi: 10.1128/MCB.00231-12

Figure Lengend Snippet: TGF-β does not inhibit BMP's ability to induce phosphorylation of Smad1/5. (A) Whole-cell extracts were prepared from MDA-MB-231 cells treated with BMP7 and/or TGF-β for the times shown. Extracts were Western blotted using antibodies against phosphorylated Smad1/5 (PSmad1/5), Smad1, and MCM6 as a loading control. The blot assays were quantitated on an ImageQuant LAS4000 mini, and the levels of PSmad1/5 relative to Smad1 are plotted on the right. (B) Same as panel A, except that nuclear extracts were assayed. (C, left side) MDA-MB-231 cells or the same cells stably expressing FLAG-ALK2 were transiently transfected with BRE-luciferase and TK-Renilla. After 24 h, cells were induced with the ligands as indicated for 8 h. Luciferase/Renilla assays were performed as in the legend to Fig. 1. (C, right side) Whole-cell extracts were prepared from the same cell lines induced for 1 h with different concentrations of BMP7. The extracts were Western blotted using antibodies against PSmad1/5, Smad1, FLAG, and MCM6 as a loading control. WT, wild type.

Article Snippet: The antibodies used were obtained from Santa Cruz Biotechnology (anti-Smad4 [B-8], anti-Smad3 [38-Q], anti-MCM7 [141.2], anti-MCM6 [C-20], and anti-Alk5 [V-22]), Sigma (anti-FLAG antibody [M2]), Life Technologies (anti-Smad1 [catalog no. 385400] and anti-Smad2 [catalog no. 511300]), Cell Signaling Technology (anti-Smad5 [catalog no. 9517], anti-phospho-Smad2 [catalog no. 3108], anti-phospho-Smad3 [catalog no. 9520S], and anti-phospho-Smad1/5/8 [catalog no. 9516]), Abcam (chromatin immunoprecipitation [ChIP] grade anti-Smad3 [ab28379] for immunoprecipitation [IP] and ChIP), Covance (RNA polymerase II [PolII; 8WG16]), and BD Biosciences (Smad2/3 [catalog no. 610843]).

Techniques: Western Blot, Stable Transfection, Expressing, Transfection, Luciferase

Journal: Molecular and Cellular Biology

Article Title: Transforming Growth Factor ? Inhibits Bone Morphogenetic Protein-Induced Transcription through Novel Phosphorylated Smad1/5-Smad3 Complexes

doi: 10.1128/MCB.00231-12

Figure Lengend Snippet: TGF-β induction leads to a loss of BMP-induced Smad1/5-Smad4 complexes and an increase in Smad1/5-Smad2/3 complexes. (A to C) MDA-MB-231 cells that were either untransfected (A and B) or transfected with nontargeting (NT) siRNA or an siRNA against Smad4 (C) were treated for 1 h with the ligands indicated. Whole-cell extracts were subjected to IP followed by Western blot assays with the antibodies shown. The lane marked “beads” correspond to an IP using beads alone. The inputs correspond to lysates prior to IP. (D) A mixture of purified recombinant PSmad1 and PSmad3 was immunoprecipitated with the antibodies shown or beads alone and then Western blotted as indicated.

Article Snippet: The antibodies used were obtained from Santa Cruz Biotechnology (anti-Smad4 [B-8], anti-Smad3 [38-Q], anti-MCM7 [141.2], anti-MCM6 [C-20], and anti-Alk5 [V-22]), Sigma (anti-FLAG antibody [M2]), Life Technologies (anti-Smad1 [catalog no. 385400] and anti-Smad2 [catalog no. 511300]), Cell Signaling Technology (anti-Smad5 [catalog no. 9517], anti-phospho-Smad2 [catalog no. 3108], anti-phospho-Smad3 [catalog no. 9520S], and anti-phospho-Smad1/5/8 [catalog no. 9516]), Abcam (chromatin immunoprecipitation [ChIP] grade anti-Smad3 [ab28379] for immunoprecipitation [IP] and ChIP), Covance (RNA polymerase II [PolII; 8WG16]), and BD Biosciences (Smad2/3 [catalog no. 610843]).

Techniques: Transfection, Western Blot, Purification, Recombinant, Immunoprecipitation