antibodies against total phospho smad1 5  (Cell Signaling Technology Inc)

Journal: International journal of molecular sciences

Article Title: Mutual Effects of Orexin and Bone Morphogenetic Proteins on Catecholamine Regulation Using Adrenomedullary Cells.

doi: 10.3390/ijms25031585

Figure Lengend Snippet: Figure 3. Interactions between orexin A and BMP during catecholamine biosynthesis via PC12 cells. (A) PC12 cells (3 × 105 cells/mL) were pretreated with orexin A (OXA; 300 nM) in DMEM containing 1% FBS and 1% HS for 48 h and were then stimulated with BMP-4 (3 ng/mL) for 1 h. The cells were lysed and then subjected to immunoblot (IB) analysis using anti-pSmad1/5/9 and anti-tSmad1 antibodies. The integrated signal density of each protein band was digitally analyzed, and the ratios of the signal intensities of pSmad/tSmad were calculated. The results are representative of those obtained from at least three independent experiments and are expressed as fold changes (n = 6). (B) Cells (3 × 105 cells/mL) were treated with BMP-4 (30 ng/mL) in DMEM containing 1% FBS and 1% HS for 24 h. Total RNAs were extracted, and the mRNA levels of OX1R and OX2R were standardized using RPL19 levels and expressed as fold changes (OX1R, n = 9; OX2R, n = 3). (C) Cells (3 × 105 cells/mL) were treated with OXA (100 nM) and/or BMP-4 (30 ng/mL) in DMEM containing 1% FBS and 1% HS for 24 h. Total RNAs were extracted, and the mRNA levels of Smad6 and Smad7 were standardized using RPL19 levels and expressed as fold changes (n = 9). (D) Cells (3 × 105 cells/mL) were treated with OXA (100 nM) in DMEM containing 1% FBS and 1% HS for 24 h. Total RNAs were extracted, and the mRNA levels of BMP receptors (ALK-2, -3 and BMPRII) were standardized using RPL19 levels and expressed as fold changes (n = 9). The results are shown as means ± SEM and were analyzed using an ANOVA with Fisher’s PLST test (A), Tukey–Kramer’s post hoc test (C) or the unpaired t-test (B–D). Values with different superscript letters are significantly different at p < 0.05. * p < 0.05 vs. the control group.

Article Snippet: The samples were then subjected to SDS-PAGE/immunoblotting analysis with primary antibodies, anti-phospho-Smad1/5/9 (pSmad1/5/9) antibodies (13,820, Cell Signaling Technology, Inc., Beverly, MA, USA) and anti-total-Smad1 (tSmad1) antibodies (9743, Cell Signaling Technology, Inc.), at 1:500 dilution and then with anti-IgG antibodies (7074, Cell Signaling Technology, Inc.) at 1:104 dilution.

Techniques: Western Blot, Control

Journal: International journal of molecular sciences

Article Title: Mutual Effects of Orexin and Bone Morphogenetic Proteins on Catecholamine Regulation Using Adrenomedullary Cells.

doi: 10.3390/ijms25031585

Figure Lengend Snippet: Figure 4. Functional interaction of the signaling between orexin A and BMP in adrenomedullary cells. Orexin A (OXA) suppressed the expression of key enzymes for catecholamine biosynthesis, including Th, Ddc and Dbh. BMP-4 downregulated the expression of Th and Ddc and enhanced the expression of Ddc in the presence and absence of OXA. OXA enhanced BMP-4-induced Smad1/5/9 phosphorylation via the downregulation of the expression of inhibitory Smad6/7 and upregulation of the expression of BMP receptors (BMPRs). BMP-4 upregulated the expression of orexin receptor type I (OX1R). In adrenomedullary cells, functional interactions between the signaling of orexin A and BMP during the regulation of catecholamine biosynthesis were demonstrated.

Article Snippet: The samples were then subjected to SDS-PAGE/immunoblotting analysis with primary antibodies, anti-phospho-Smad1/5/9 (pSmad1/5/9) antibodies (13,820, Cell Signaling Technology, Inc., Beverly, MA, USA) and anti-total-Smad1 (tSmad1) antibodies (9743, Cell Signaling Technology, Inc.), at 1:500 dilution and then with anti-IgG antibodies (7074, Cell Signaling Technology, Inc.) at 1:104 dilution.

Techniques: Functional Assay, Expressing, Phospho-proteomics

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B is superior to ACVR2A in eliciting ALK2-R206H-mediated transcriptional activation of the SMAD1/5/8 pathway. U2OS cells were co-transfected with BRE-Luc and pRL-TK, together with HA-ALK2-R206H or HA-ALK2-WT (alone or together with myc-ACVR2A, myc-ACVR2B, or myc-ACVR2B-KD). These constructs were replaced by empty vector for control samples. After 17 h, cells were starved without serum (5 h) and stimulated (or not; control) with ActA (2 nM, 19 h). Relative Luminescence Units (RLU) are expressed as mean fold induction ± SEM (n = 4 independent experiments). The results were normalized for transfection efficiency using Renilla luminescence by the DLR luminescence assay. The value in untreated, unstimulated cells was taken as 1. The cell-surface levels of the tagged receptors were not altered by the co-expressed receptors . Asterisks show significant differences between the pairs indicated by the brackets, using one-way ANOVA and Bonferroni post hoc test (**, p < 1 × 10 −3 ; ***, p < 5 × 10 −4 ; ****, p < 10 −4 ; n.s. = not significant).

Article Snippet: Rabbit antibodies to detect phospho (p) SMAD1/5/8 (cat. #13820) or total (t) SMAD1/5/8 (cat. #6944) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and mouse anti-β-actin (cat. #0869100-CF) was obtained from MP Biomedicals (Solon, OH, USA).

Techniques: Activation Assay, Transfection, Construct, Plasmid Preparation, Luminescence Assay

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: Model for the recruitment of ALK2-R206H into homomeric clusters by ACVR2A/B and its dependence on the dimeric nature of the type II receptors. ( A ) Homodimerization state of the singly-expressed receptors and the dependence on ActA. ACVR2B (red) forms stable homodimers already without ActA, which are enhanced by the ligand (thicker black arrow). ACVR2A (green) requires ActA (orange) to form homodimers. ALK2-WT (light blue) forms homodimers, while ALK2-R206H (dark blue) does not, and both are unaffected by ActA. ( B ) Effect of complex formation with ACVR2A or ACVR2B on the homomeric clustering of ALK2-R206H. The recruitment of the mainly monomeric ALK2-R206H into clusters by the type II receptor depends on the extent of homodimerization of the type II receptor. Thus, the largely dimeric ACVR2B can induce ALK2-R206H clustering already without ligand, while ActA enhances this effect due to increasing ACVR2B homomeric complex formation and its heteromeric interactions with ALK2-R206H. On the other hand, ACVR2A cannot induce clustering of ALK2-R206H, as both receptors are mainly monomeric in the absence of ligand. Upon binding of ActA, ACVR2A forms homodimers and can then induce clustering of ALK2-R206H. The homomeric clustering of ALK2-R206H leads to aberrant signaling to SMAD1/5/8 without a need for phosphorylation by the type II receptor.

Article Snippet: Rabbit antibodies to detect phospho (p) SMAD1/5/8 (cat. #13820) or total (t) SMAD1/5/8 (cat. #6944) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and mouse anti-β-actin (cat. #0869100-CF) was obtained from MP Biomedicals (Solon, OH, USA).

Techniques: Binding Assay