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antibodies against smad1 6944 (Cell Signaling Technology Inc)

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Brand: Cell Signaling Technology Inc
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Cell Signaling Technology Inc antibodies against smad1 6944

( A ) Western blot analysis detecting C-terminal phosphorylated <t>SMAD1/5,</t> total SMAD1, and vinculin. ( B ) RT-qPCR analysis of the BMP target genes ID1 and ID3 ( n = 3 per group). Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, by two-way ANOVA with Tukey’s multiple comparisons test. Figure 5—figure supplement 1—source data 1. Original files for western blot analysis displayed in . Figure 5—figure supplement 1—source data 2. PDF file containing original western blots for , indicating the relevant bands and treatments.

Antibodies Against Smad1 6944, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "PI3Kα inhibition blocks osteochondroprogenitor specification and the hyper-inflammatory response to prevent heterotopic ossification"

Article Title: PI3Kα inhibition blocks osteochondroprogenitor specification and the hyper-inflammatory response to prevent heterotopic ossification

Journal: eLife

doi: 10.7554/eLife.91779

( A ) Western blot analysis detecting C-terminal phosphorylated SMAD1/5, total SMAD1, and vinculin. ( B ) RT-qPCR analysis of the BMP target genes ID1 and ID3 ( n = 3 per group). Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, by two-way ANOVA with Tukey’s multiple comparisons test. Figure 5—figure supplement 1—source data 1. Original files for western blot analysis displayed in . Figure 5—figure supplement 1—source data 2. PDF file containing original western blots for , indicating the relevant bands and treatments.

Figure Legend Snippet: ( A ) Western blot analysis detecting C-terminal phosphorylated SMAD1/5, total SMAD1, and vinculin. ( B ) RT-qPCR analysis of the BMP target genes ID1 and ID3 ( n = 3 per group). Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, by two-way ANOVA with Tukey’s multiple comparisons test. Figure 5—figure supplement 1—source data 1. Original files for western blot analysis displayed in . Figure 5—figure supplement 1—source data 2. PDF file containing original western blots for , indicating the relevant bands and treatments.

Techniques Used: Western Blot, Quantitative RT-PCR

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Journal: eLife

Article Title: PI3Kα inhibition blocks osteochondroprogenitor specification and the hyper-inflammatory response to prevent heterotopic ossification

doi: 10.7554/eLife.91779

Figure Lengend Snippet: ( A ) Western blot analysis detecting C-terminal phosphorylated SMAD1/5, total SMAD1, and vinculin. ( B ) RT-qPCR analysis of the BMP target genes ID1 and ID3 ( n = 3 per group). Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, by two-way ANOVA with Tukey’s multiple comparisons test. Figure 5—figure supplement 1—source data 1. Original files for western blot analysis displayed in . Figure 5—figure supplement 1—source data 2. PDF file containing original western blots for , indicating the relevant bands and treatments.

Article Snippet: Primary antibodies against SMAD1 (Cell Signaling, 6944), p-SMAD1/5 (Cell Signaling, 9516), and vinculin (Sigma, V9131) were used at 1:1000 dilution in 5% BSA in TBST.

Techniques: Western Blot, Quantitative RT-PCR

Fig. 4 Deletion of nesfatin-1 diminishes BMP-2 osteogenic signaling. (A) Quantitative RT-PCR analysis of RUNX2, SOX9, MSX2, α-SMA in VSMCs trans fected with scramble vector or nesfatin-1 overexpression plasmid in the presence or absence of soluble BMPR-IA (1 mg/L). (B) Western blot analysis of Smad, RUNX2 and MSX2. (C) Quantification of calcium deposition. (D) ALP activity. (E) Acetylation level of RUNX2. (F) RUNX2 protein expression in the nucleus and cytoplasm. (G) Immunofluorescence staining of RUNX2. Scale bar, 25 μm. (H) The phosphorylation level of HDAC4. (I) HDAC4 protein expres sion in the nucleus and cytoplasm. (J) Immunofluorescence staining of HDAC4. Scale bar, 25 μm. Differences between groups were assessed with ANOVA followed by Bonferroni post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group. n = 6

Journal: Cell communication and signaling : CCS

Article Title: Nesfatin-1 enhances vascular smooth muscle calcification through facilitating BMP-2 osteogenic signaling.

doi: 10.1186/s12964-024-01873-7

Figure Lengend Snippet: Fig. 4 Deletion of nesfatin-1 diminishes BMP-2 osteogenic signaling. (A) Quantitative RT-PCR analysis of RUNX2, SOX9, MSX2, α-SMA in VSMCs trans fected with scramble vector or nesfatin-1 overexpression plasmid in the presence or absence of soluble BMPR-IA (1 mg/L). (B) Western blot analysis of Smad, RUNX2 and MSX2. (C) Quantification of calcium deposition. (D) ALP activity. (E) Acetylation level of RUNX2. (F) RUNX2 protein expression in the nucleus and cytoplasm. (G) Immunofluorescence staining of RUNX2. Scale bar, 25 μm. (H) The phosphorylation level of HDAC4. (I) HDAC4 protein expres sion in the nucleus and cytoplasm. (J) Immunofluorescence staining of HDAC4. Scale bar, 25 μm. Differences between groups were assessed with ANOVA followed by Bonferroni post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group. n = 6

Article Snippet: The following antibodies were used: anti-nesfatin-1 antibody (#26712-1-AP, Proteintech), anti-RUNX2 antibody (#12556S, Cell Signaling Technology or 82636-2-RR, Proteintech), anti-BMP-2 antibody (#ER80602, Huabio), anti-α-SMA antibody (#14395-1- AP, Proteintech), anti-SM22α antibody (#10493-1-AP, Proteintech), anti-HDAC4 antibody (#7628S, Cell Signaling Technology), anti-p-Smad antibody (#13820, CST), anti-T-Smad antibody (#6944, CST), anti-Msx2 antibody (#orb415625, Biorbyt), anti-Lamin B1 (#12987-1-AP, Proteintech), anti-HA (81290-1-RR, Proteintech), anti-Flag antibody (66008-4-Ig, Proteintech), anti-β-actin (#4970, CST), anti-His antibody, (#66005-1-Ig Proteintech), antiSTAT3 (#10253-2-AP, Proteintech), anti-HDAC4 antibody (#7628, CST), anti-p-HDAC4 antibody (Ser246, #bs-10325R, Bioss), anti-Ub antibody (#10201-2-AP, Proteintech), anti-GAPDH antibody (#60004-1-Ig, Proteintech), Anti-Acetylated-Lysine antibody (#9441, CST), anti-SYTL4 antibody (#12128-1-AP, Proteintech), antiSTAT3 antibody (#9139, CST), anti-β-tubulin (#M056136, Boster), HRP-linked anti-rabbit IgG (SA00001-2, Proteintech), HRP-linked anti-mouse IgG (SA00001-1, Proteintech).

Techniques: Quantitative RT-PCR, Plasmid Preparation, Over Expression, Western Blot, Activity Assay, Expressing, Immunofluorescence, Staining, Phospho-proteomics

FIGURE 4 Effect of OSM on the BMP‐4‐induced phosphorylation of SMAD1/5/9 in MC3T3‐E1 cells. The cultured cells were pretreated with 30 ng/mL of OSM or vehicle for 60 min, and then stimulated by 30 ng/mL of BMP‐4 or vehicle for 45 min. The cell extracts were then subjected to SDS‐PAGE and subsequent Western blot analysis with antibodies against phospho‐specific SMAD1/5/9, SMAD1, and GAPDH. The histogram shows the quantitative representations of phosphorylated SMAD1/5/9 levels after normalization with respect to SMAD1/5/9 (upper panel) or GAPDH (lower panel) obtained from laser densitometric analysis. The levels were expressed as the fold increase with respect to the basal levels presented in lane 1. Each value represents the mean ± SEM of triplicate determinations obtained from three independent cell preparations. *p < .05 versus control; N.S., not significant. BMP‐4, bone morphogenetic protein‐4; OSM, oncostatin M; SDS, sodium dodecyl sulfate; SEM, standard error of the mean.

Journal: Cell biochemistry and function

Article Title: Oncostatin M suppresses bone morphogenetic protein-4-induced osteoprotegerin synthesis in MC3T3-E1 osteoblast-like cells: p70 S6 kinase attenuation.

doi: 10.1002/cbf.4068

Figure Lengend Snippet: FIGURE 4 Effect of OSM on the BMP‐4‐induced phosphorylation of SMAD1/5/9 in MC3T3‐E1 cells. The cultured cells were pretreated with 30 ng/mL of OSM or vehicle for 60 min, and then stimulated by 30 ng/mL of BMP‐4 or vehicle for 45 min. The cell extracts were then subjected to SDS‐PAGE and subsequent Western blot analysis with antibodies against phospho‐specific SMAD1/5/9, SMAD1, and GAPDH. The histogram shows the quantitative representations of phosphorylated SMAD1/5/9 levels after normalization with respect to SMAD1/5/9 (upper panel) or GAPDH (lower panel) obtained from laser densitometric analysis. The levels were expressed as the fold increase with respect to the basal levels presented in lane 1. Each value represents the mean ± SEM of triplicate determinations obtained from three independent cell preparations. *p < .05 versus control; N.S., not significant. BMP‐4, bone morphogenetic protein‐4; OSM, oncostatin M; SDS, sodium dodecyl sulfate; SEM, standard error of the mean.

Article Snippet: The proteins were then incubated with the primary antibody as previously indicated.16 Phospho‐specific SMAD1/5/9 antibodies (cat. no. 13820), SMAD1 antibodies (cat. no. 6944), phospho‐specific p38 MAPK antibodies (cat. no. 4511), p38 MAPK antibodies (cat. no. 9212), phospho‐specific p70 S6 kinase antibodies (cat. no. 9208), or p70 S6 kinase antibodies (cat. no. 9202) obtained from Cell SignalingTechnology Inc., or GAPDH antibodies (cat. no. 60004‐1‐lg) obtained from Proteintech Group Inc. were used as primary antibodies.

Techniques: Phospho-proteomics, Cell Culture, SDS Page, Western Blot, Control

FIGURE 2 Bovine Smad4 is less effective than murine Smad4 for hepcidin reporter expression. (A) HepG2 cells or BH5 cells were treated with BMP6 (2 nM) or TGF-β1 (200 pM) for 12 h. The expression levels of hepcidin, Smad6, and Smad7 were evaluated by RT–qPCR. **p < .01 vs. the control cells. Mean ± SE (n = 4). (B and E) HepG2 cells (B) or SW480.7 cells (E) were treated with BMP6 (0, 0.125, 0.25, 0.5, 1, or 2 nM) for 1 h. Phosphorylated Smad1/5/8 and Smad1 levels were evaluated by western blot analysis. (C and F) HepG2 cells (C) or SW480.7 cells (F) were transfected with the indicated reporter, Smad4 expression vector from the indicated species, and expression vector encoding β-galactosidase and then treated with BMP6 (0, 0.125, 0.25, 0.5, 1, or 2 nM). (D and G) HepG2 cells (D) or SW480.7 cells (G) were transfected with the indicated reporter, Smad1 or Smad4 from the indicated species, and an expression vector encoding β-galactosidase. (C, D, F, and G) Luciferase activity was normalized to that of β-galactosidase, and the relative expression in the control cells transfected with mHepc-luc was set to 1. The number on the bar indicates the fold change relative to the normalized luciferase activity in the control cells transfected with each reporter. m: expression vector encoding murine Smad. b: Expression vector encoding bovine Smad. Mean ± SE (n = 3).

Journal: The FASEB Journal

Article Title: Weak response of bovine hepcidin induction to iron through decreased expression of Smad4

doi: 10.1096/fj.202301186rr

Figure Lengend Snippet: FIGURE 2 Bovine Smad4 is less effective than murine Smad4 for hepcidin reporter expression. (A) HepG2 cells or BH5 cells were treated with BMP6 (2 nM) or TGF-β1 (200 pM) for 12 h. The expression levels of hepcidin, Smad6, and Smad7 were evaluated by RT–qPCR. **p < .01 vs. the control cells. Mean ± SE (n = 4). (B and E) HepG2 cells (B) or SW480.7 cells (E) were treated with BMP6 (0, 0.125, 0.25, 0.5, 1, or 2 nM) for 1 h. Phosphorylated Smad1/5/8 and Smad1 levels were evaluated by western blot analysis. (C and F) HepG2 cells (C) or SW480.7 cells (F) were transfected with the indicated reporter, Smad4 expression vector from the indicated species, and expression vector encoding β-galactosidase and then treated with BMP6 (0, 0.125, 0.25, 0.5, 1, or 2 nM). (D and G) HepG2 cells (D) or SW480.7 cells (G) were transfected with the indicated reporter, Smad1 or Smad4 from the indicated species, and an expression vector encoding β-galactosidase. (C, D, F, and G) Luciferase activity was normalized to that of β-galactosidase, and the relative expression in the control cells transfected with mHepc-luc was set to 1. The number on the bar indicates the fold change relative to the normalized luciferase activity in the control cells transfected with each reporter. m: expression vector encoding murine Smad. b: Expression vector encoding bovine Smad. Mean ± SE (n = 3).

Article Snippet: BMP6 was purchased from GeneTex (Irvine, CA, USA); TGF- β1 was obtained from R&D Systems (Minneapolis, MN, USA); mouse monoclonal antibody against c- Myc antibody (9E10, sc- 40), rabbit polyclonal antibody against Smad4 (H- 552, sc- 7154), and mouse monoclonal antibody against Smad4 (B- 8, sc- 7966) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit polyclonal antibody against β- actin (#4967), rabbit monoclonal antibody against phosphorylated Smad1/5/8 (D5B10, #13820), rabbit monoclonal antibody against Smad1 (D59D7, #6944), and rabbit monoclonal antibody against Smad2/3 (D7G7, #8685) were purchased from Cell Signaling Technology (Danvers, MA, USA); rabbit monoclonal antibody against Smad1 (EP565Y, ab33902) was purchased from Abcam (Cambridge, UK); polyethylenimine (PEI) Max reagent was purchased from Polysciences (Warrington, PA, USA); and Lipofectamine RNAiMax and DNase I were purchased from Thermo Fisher Scientific (Waltham, MA, USA); Precision Plus Protein Standard was purchased from Bio- Rad Laboratories (Hercules, CA, USA); and Excelband All Blue Broad Range Protein Marker was purchased from SMOBIO Technology (Hsinchu City, TW).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay

FIGURE 3 Bovine Smad4 mediates BMP signaling with less potency than murine Smad4. (A, C, and D) SW480.7 cells were transfected with plasmid constructs encoding Smad1 or Smad4 from the indicated species, with the indicated reporter and expression vector encoding β-galactosidase. (B) HepG2 cells were transfected with the indicated siRNA, followed by plasmid constructs encoding Smad4 from the indicated species, with mHepc-luc and an expression vector encoding β-galactosidase. Luciferase activity was normalized to that of β- galactosidase, and the relative expression in the control cells transfected with mHepc-luc (A and B) or BRE-luc (C and D) was set to 1. The number on the bar indicates the fold change relative to the normalized luciferase activity in the control cells transfected with each reporter. Mean ± SE (n = 3).

Journal: The FASEB Journal

Article Title: Weak response of bovine hepcidin induction to iron through decreased expression of Smad4

doi: 10.1096/fj.202301186rr

Figure Lengend Snippet: FIGURE 3 Bovine Smad4 mediates BMP signaling with less potency than murine Smad4. (A, C, and D) SW480.7 cells were transfected with plasmid constructs encoding Smad1 or Smad4 from the indicated species, with the indicated reporter and expression vector encoding β-galactosidase. (B) HepG2 cells were transfected with the indicated siRNA, followed by plasmid constructs encoding Smad4 from the indicated species, with mHepc-luc and an expression vector encoding β-galactosidase. Luciferase activity was normalized to that of β- galactosidase, and the relative expression in the control cells transfected with mHepc-luc (A and B) or BRE-luc (C and D) was set to 1. The number on the bar indicates the fold change relative to the normalized luciferase activity in the control cells transfected with each reporter. Mean ± SE (n = 3).

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Luciferase, Activity Assay, Control

FIGURE 4 The nucleotide sequence of the MH1 domain determines the reporter activity of hepcidin and activation of the BMP pathway. (A,B) SW480.7 cells were transfected with the plasmid construct encoding murine Smad1, the indicated chimeric constructs of murine or bovine Smad4, reporter plasmid with mHepc-luc (A) or BRE-luc (B), and expression vector encoding β-galactosidase. Smad4 is divided into three parts: the MH1 domain, linker region, and MH2 domain. The indicated chimeric Smad4, for example, MMB, consists of the murine MH1 domain, murine linker region, and bovine MH2 domain. Luciferase activity was normalized to that of β-galactosidase, and the relative expression in the control cells transfected with mHepc-luc (A) or BRE-luc (B) was set to 1. Mean ± SE (n = 3).

Journal: The FASEB Journal

Article Title: Weak response of bovine hepcidin induction to iron through decreased expression of Smad4

doi: 10.1096/fj.202301186rr

Figure Lengend Snippet: FIGURE 4 The nucleotide sequence of the MH1 domain determines the reporter activity of hepcidin and activation of the BMP pathway. (A,B) SW480.7 cells were transfected with the plasmid construct encoding murine Smad1, the indicated chimeric constructs of murine or bovine Smad4, reporter plasmid with mHepc-luc (A) or BRE-luc (B), and expression vector encoding β-galactosidase. Smad4 is divided into three parts: the MH1 domain, linker region, and MH2 domain. The indicated chimeric Smad4, for example, MMB, consists of the murine MH1 domain, murine linker region, and bovine MH2 domain. Luciferase activity was normalized to that of β-galactosidase, and the relative expression in the control cells transfected with mHepc-luc (A) or BRE-luc (B) was set to 1. Mean ± SE (n = 3).

Techniques: Sequencing, Activity Assay, Activation Assay, Transfection, Plasmid Preparation, Construct, Expressing, Luciferase, Control

FIGURE 5 Protein level of bovine Smad4 transfected with its expression vector is lower than that of murine Smad4. (A) SW480.7 cells were transfected with plasmid constructs encoding 6Myc-Smad4 at 0.0625, 0.125, 0.25, 0.5, or 1.0 μg/well in a 12-well plate. (B–D) HepG2 cells or SW480.7 cells were transfected with the indicated 6Myc-Smad at 0.5 or 1.0 μg/well in a 12-well plate. The expression of transfected Smad was evaluated by western blot analysis. β-Actin was used as the loading control. (B) m1, b1, m4, and m4: murine Smad1, bovine Smad1, murine Smad4, and bovine Smad4, respectively. (C) m, b*, b, and m*: murine Smad4, murine Smad4 mutated to bovine Smad4 at the amino acid level, bovine Smad4, and bovine Smad4 mutated to murine Smad4 at the amino acid level, respectively. (D) Smad4 is divided into three parts, the MH1 domain, linker region, and MH2 domain, and the indicated chimeric Smad4, for example, MMB, consists of the murine MH1 domain, murine linker region, and bovine MH2 domain.

Journal: The FASEB Journal

Article Title: Weak response of bovine hepcidin induction to iron through decreased expression of Smad4

doi: 10.1096/fj.202301186rr

Figure Lengend Snippet: FIGURE 5 Protein level of bovine Smad4 transfected with its expression vector is lower than that of murine Smad4. (A) SW480.7 cells were transfected with plasmid constructs encoding 6Myc-Smad4 at 0.0625, 0.125, 0.25, 0.5, or 1.0 μg/well in a 12-well plate. (B–D) HepG2 cells or SW480.7 cells were transfected with the indicated 6Myc-Smad at 0.5 or 1.0 μg/well in a 12-well plate. The expression of transfected Smad was evaluated by western blot analysis. β-Actin was used as the loading control. (B) m1, b1, m4, and m4: murine Smad1, bovine Smad1, murine Smad4, and bovine Smad4, respectively. (C) m, b*, b, and m*: murine Smad4, murine Smad4 mutated to bovine Smad4 at the amino acid level, bovine Smad4, and bovine Smad4 mutated to murine Smad4 at the amino acid level, respectively. (D) Smad4 is divided into three parts, the MH1 domain, linker region, and MH2 domain, and the indicated chimeric Smad4, for example, MMB, consists of the murine MH1 domain, murine linker region, and bovine MH2 domain.

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Western Blot, Control

FIGURE 6 mRNA stability of bovine Smad4 is lower than that of murine Smad4. (A) SW480.7 cells were transfected with plasmid constructs encoding Smad4 (0.5, 1.0, or 2.0 μg/well of a 12-well plate). The expression levels of transfected Smad4 mRNA were evaluated by RT–qPCR. The levels in the cells transfected with 0.5 μg of murine Smad4 were defined as 1. The results of two-way ANOVA are shown. Mean ± SE (n = 3). (B) HepG2 and BH5 cells were treated with actinomycin D (5 μg/mL) for the indicated periods. The mRNA levels of Smad were evaluated by RT–qPCR, and the mRNA level normalized to the amount of total RNA in each cell at 0 h of actinomycin D treatment was set at 1. The data are expressed as the mean ± SE (n = 4). (C) Expression of the endogenous Smad protein was examined in HepG2, Hepa1-6, SW480.7, BH4, BH5, and MDBK cells by western blot analysis using anti-Smad1 antibody (D59D7), anti-Smad2/3 antibody (D7G7), anti- Smad4 antibody (H-552), and anti-β-actin antibody. β-Actin was used as the loading control.

Journal: The FASEB Journal

Article Title: Weak response of bovine hepcidin induction to iron through decreased expression of Smad4

doi: 10.1096/fj.202301186rr

Figure Lengend Snippet: FIGURE 6 mRNA stability of bovine Smad4 is lower than that of murine Smad4. (A) SW480.7 cells were transfected with plasmid constructs encoding Smad4 (0.5, 1.0, or 2.0 μg/well of a 12-well plate). The expression levels of transfected Smad4 mRNA were evaluated by RT–qPCR. The levels in the cells transfected with 0.5 μg of murine Smad4 were defined as 1. The results of two-way ANOVA are shown. Mean ± SE (n = 3). (B) HepG2 and BH5 cells were treated with actinomycin D (5 μg/mL) for the indicated periods. The mRNA levels of Smad were evaluated by RT–qPCR, and the mRNA level normalized to the amount of total RNA in each cell at 0 h of actinomycin D treatment was set at 1. The data are expressed as the mean ± SE (n = 4). (C) Expression of the endogenous Smad protein was examined in HepG2, Hepa1-6, SW480.7, BH4, BH5, and MDBK cells by western blot analysis using anti-Smad1 antibody (D59D7), anti-Smad2/3 antibody (D7G7), anti- Smad4 antibody (H-552), and anti-β-actin antibody. β-Actin was used as the loading control.

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Quantitative RT-PCR, Western Blot, Control

Figure 4. Three-dimensional structural prediction of ACVRL1 interaction with SMAD1 and BMPR2. ACVRL1 kinase domain (green; aa 195-503), BMPR2 kinase domain (cyan; aa 197-512) and SMAD1 MH2 domain (magenta; aa 271-465) were applied to the structural prediction using ColabFold. (A) The predicted structure of the ACVRL1-BMPR2-SMAD1 trimer viewed from two different directions. A top view of the left image is on the right. (B) ACVRL1-SMAD1 dimer structure (left) with an enlarged view of the dotted box (middle). The ﬁgure on the right shows the area in the middle ﬁgure viewed from another direction. Side chains of ACVRL1 D263 and T265, that of SMAD1 N280 and a main chain of ACVRL1 A199 are depicted. In the ACVRL1-SMAD1 interface shown on the right, the interaction between ACVRL1 D263 in the L45 loop and SMAD1 N280 was predicted. Intramolecular interaction between ACVRL1 D263 and A199 was also indicated. D263 and T265 of ACVRL1 are localized in the same β sheet. (C) ACVRL1-BMPR2 dimer structure (left) with an enlarged view of the dotted box (middle). The ﬁgure on the right depicts the image in the middle ﬁgure viewed from another direction. In the ACVRL1-BMPR2 interface shown on the right, the interaction between ACVRL1 R484 in the NANDOR domain with BMPR2 D482 and D485 was predicted. On the other hand, ACVRL1 R479 did not show direct interaction with BMPR2. Carbon, oxygen and nitrogen atoms are represented by gray, red and blue, respectively, and hydrogen atoms are omitted for clarity. Dotted lines indicate possible amino acid interaction with the distance less than 4 Å.

Journal: Journal of clinical medicine

Article Title: Computational and Experimental Analyses for Pathogenicity Prediction of ACVRL1 Missense Variants in Hereditary Hemorrhagic Telangiectasia.

doi: 10.3390/jcm12155002

Figure Lengend Snippet: Figure 4. Three-dimensional structural prediction of ACVRL1 interaction with SMAD1 and BMPR2. ACVRL1 kinase domain (green; aa 195-503), BMPR2 kinase domain (cyan; aa 197-512) and SMAD1 MH2 domain (magenta; aa 271-465) were applied to the structural prediction using ColabFold. (A) The predicted structure of the ACVRL1-BMPR2-SMAD1 trimer viewed from two different directions. A top view of the left image is on the right. (B) ACVRL1-SMAD1 dimer structure (left) with an enlarged view of the dotted box (middle). The ﬁgure on the right shows the area in the middle ﬁgure viewed from another direction. Side chains of ACVRL1 D263 and T265, that of SMAD1 N280 and a main chain of ACVRL1 A199 are depicted. In the ACVRL1-SMAD1 interface shown on the right, the interaction between ACVRL1 D263 in the L45 loop and SMAD1 N280 was predicted. Intramolecular interaction between ACVRL1 D263 and A199 was also indicated. D263 and T265 of ACVRL1 are localized in the same β sheet. (C) ACVRL1-BMPR2 dimer structure (left) with an enlarged view of the dotted box (middle). The ﬁgure on the right depicts the image in the middle ﬁgure viewed from another direction. In the ACVRL1-BMPR2 interface shown on the right, the interaction between ACVRL1 R484 in the NANDOR domain with BMPR2 D482 and D485 was predicted. On the other hand, ACVRL1 R479 did not show direct interaction with BMPR2. Carbon, oxygen and nitrogen atoms are represented by gray, red and blue, respectively, and hydrogen atoms are omitted for clarity. Dotted lines indicate possible amino acid interaction with the distance less than 4 Å.

Article Snippet: 2023, 12, 5002 4 of 18 (Cell Signaling Technology, Danvers, MA, USA), SMAD1 antibody #6944 (Cell Signaling Technology), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody MAB374 (Millipore, Merck, Burlington, NJ, USA).

Techniques: Structural Proteomics

Figure 6. Signal transduction capacity of D176Y, R200G, V205G and D235Y variants. (A) D176Y, R200G, V205G and D235Y variants showed BMP9 dose dependency similar to wild-type (WT) ACVRL1 in BRE-luciferase assays. Results are expressed as fold induction over the value obtained for the cells expressing wild-type ACVRL1 with the vehicle treatment. * p < 0.05; ** p < 0.01; *** p < 0.001 (Dunnett’s test). (B,C) The cells expressing D176Y, R200G, V205G and D235Y variants showed BMP9- induced transcriptional activity in ID1- or BMPR2-luciferase assays. Results are expressed as fold induction over the values obtained for vehicle-treated wild-type ACVRL1. *** p < 0.001 (Tukey’s test). (D) The cells expressing D176Y, R200G, V205G and D235Y variants showed SMAD1/5/9 phospho- rylation in response to the BMP9 treatment, which was comparable to those expressing wild-type ACVRL1. Note that vector-transfected cells did not show signiﬁcant SMAD1/5/9 phosphorylation. Western blot analysis.

Journal: Journal of clinical medicine

Article Title: Computational and Experimental Analyses for Pathogenicity Prediction of ACVRL1 Missense Variants in Hereditary Hemorrhagic Telangiectasia.

doi: 10.3390/jcm12155002

Figure Lengend Snippet: Figure 6. Signal transduction capacity of D176Y, R200G, V205G and D235Y variants. (A) D176Y, R200G, V205G and D235Y variants showed BMP9 dose dependency similar to wild-type (WT) ACVRL1 in BRE-luciferase assays. Results are expressed as fold induction over the value obtained for the cells expressing wild-type ACVRL1 with the vehicle treatment. * p < 0.05; ** p < 0.01; *** p < 0.001 (Dunnett’s test). (B,C) The cells expressing D176Y, R200G, V205G and D235Y variants showed BMP9- induced transcriptional activity in ID1- or BMPR2-luciferase assays. Results are expressed as fold induction over the values obtained for vehicle-treated wild-type ACVRL1. *** p < 0.001 (Tukey’s test). (D) The cells expressing D176Y, R200G, V205G and D235Y variants showed SMAD1/5/9 phospho- rylation in response to the BMP9 treatment, which was comparable to those expressing wild-type ACVRL1. Note that vector-transfected cells did not show signiﬁcant SMAD1/5/9 phosphorylation. Western blot analysis.

Techniques: Transduction, Luciferase, Expressing, Activity Assay, Plasmid Preparation, Transfection, Phospho-proteomics, Western Blot

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