Journal: Animals : an Open Access Journal from MDPI

Article Title: Effect of the TGF-β/BMP Signaling Pathway on the Proliferation of Yak Pulmonary Artery Smooth Muscle Cells under Hypoxic Conditions

doi: 10.3390/ani14142072

Figure Lengend Snippet: Impact of hypoxia on BMP/TGF-β signaling pathway-related protein expression and proliferation in yak PASMCs. ( A – C ) Western blot analysis of HIF-1α and HIF-2α protein expression under hypoxic conditions, with a densitometric analysis performed using ImageJ. ( D – G ) Western blot analysis of TGF-β/Smad2/3 signaling pathway-related protein expression under hypoxia conditions, with a densitometric analysis performed using ImageJ. ( H – J ) Western blot analysis of BMP/Smad1/5 signaling pathway-related protein expression under hypoxic conditions, with a densitometric analysis performed using ImageJ. ( K – M ) Western blot analysis of BCL-2 and PCNA protein expression levels under hypoxic conditions, with a densitometric analysis performed using ImageJ. ** p < 0.01, * 0.01 < p < 0.05, ns indicates insignificant difference.

Article Snippet: The membrane was blocked at room temperature with 50 g·L −1 non-fat dry milk powder for 3 h. Then, the samples were incubated overnight at 4 °C with primary antibodies for HIF-1α (1:1000; ab16066, Abcam, Cambridge, MA, USA), HIF-2α (1:800; AF6362, Affinity, Jiangsu, China), TGF-β1 (1:500; bs-0086R, Bioss, Beijing, China), Smad2 (1:500; bs-0718R, Bioss, Beijing, China), Smad3 (1:500; bs-8853R, Bioss, Beijing, China), phosphorylated Smad2 (P-Smad2) (1:400; bs-8853R, Bioss, Beijing, China), phosphorylated Smad3 (P-Smad3) (1:400; bs-3425R, Bioss, Beijing, China), BMPR2 (1:500, bs-4237R; Bioss, Beijing, China), Smad1/5 (1:500; AF0614, Affinity, Jiangsu, China), phosphorylated Smad1/5 (P-Smad1/5) (1:400; bs-3418R, Bioss, Beijing, China), PCNA (1:500; bs-2006R, Bioss, Beijing, China), and BCL-2 (1:500; bs-0032R, Bioss, Beijing, China).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Tbx1 Regulates the BMP-Smad1 Pathway in a Transcription Independent Manner

doi: 10.1371/journal.pone.0006049

Figure Lengend Snippet: a) Western blot analysis of affinity-purified nuclear extracts from P19-Tbx1-PA (lane 1) and P19-PA (lane 3) cells, compared non-purified nuclear extracts of P19-Tbx1-PA cells (lane 2), using an antibody anti-Phospho-SMAD1/5/8. b) Western blot analyses of reciprocal coimmunoprecipitation (IP) experiments using the antibodies indicated. NIH3T3 cells were co-transfected transiently with Tbx1-cmyc and Smad1-Flag expression vectors. c) Western blot analyses of nuclear extracts from wild type E9.5 mouse embryos immunoprecipitated with an anti-Tbx1 antibody (lanes 1 and 2) or with anti-rabbit IgG (lane 3) and revealed with an anti-SMAD1 antibody (lane 1 and 3) and an anti-Phospho-SMAD1/5/8 antibody (lane 2). d) Immunoblotting with anti-Smad1 antibody of nuclear extracts of wild type mouse embryos (E9.5) coimmunoprecipitated with anti-Tbx1 (NE-IP), total nuclear extracts (NE), total cytoplasmic extracts (CE), and cytoplasmic extracts coimminoprecipitated with anti-Tbx1 (CE-IP). e) Coimmunoprecipitation of WT TBX1, TBX1 G145R , TBX1 F148Y , TBX1 G310S and Flag-SMAD1 transiently transfected in NIH 3T3 cells. The TBX1 G145R and TBX1 F148Y mutants physically interact with Smad1, while TBX1 G310S is unable to bind Smad1 indicating that Glycine 310 is important for this interaction. f) Luciferase assay using Cos7 cells transfected with the SMAD-responsive reporter NTK-tetramer-luc. Transfected SMAD1 activates the reporter while increasing amounts (5 to 100 ng) of co-transfected TBX1 suppresses it.

Article Snippet: The anti-Tbx1 antibody was obtained from Zymed Laboratories; the anti-P-Smad1 (Ser463/Ser465) from Chemicon; The anti Smad1, P-Smad2 (Ser465/Ser467), Smad4, Smad5, and Smad6 where all purchased from Cell Signaling.

Techniques: Western Blot, Affinity Purification, Purification, Transfection, Expressing, Immunoprecipitation, Luciferase

Journal: PLoS ONE

Article Title: Tbx1 Regulates the BMP-Smad1 Pathway in a Transcription Independent Manner

doi: 10.1371/journal.pone.0006049

Figure Lengend Snippet: a) A luciferase assay showing the inability of the TBX1 G145R mutant to transactivate a T-box reporter construct in Jeg3 cells. Error bars indicate the standard error mean. b) A luciferase assay with a SMAD reporter showing that the mutant is capable of suppressing SMAD transactivation. c) Western blot analyses of nuclear extracts from C2C12 transfected with Tbx1 and SMAD1 -flag expression vectors (as indicated). The top two rows are samples immunoprecipitated with an anti-flag antibody. The bottom two rows are non-immunoprecipitated nuclear extracts from the same samples. Note the strong reduction of Smad4 co-immunoprecipitated with Smad1 in the presence of transfected TBX1.

Article Snippet: The anti-Tbx1 antibody was obtained from Zymed Laboratories; the anti-P-Smad1 (Ser463/Ser465) from Chemicon; The anti Smad1, P-Smad2 (Ser465/Ser467), Smad4, Smad5, and Smad6 where all purchased from Cell Signaling.

Techniques: Luciferase, Mutagenesis, Construct, Western Blot, Transfection, Expressing, Immunoprecipitation