Journal: International Journal of Molecular Sciences

Article Title: αvβ3 Integrin as a Link between the Development of Fibrosis and Thyroid Hormones in Systemic Sclerosis

doi: 10.3390/ijms24108927

Figure Lengend Snippet: List of antibodies.

Article Snippet: Elastin , Rabbit , Polyclonal Ab , Proteintech , 15257-1 , 1:2500 , BSA 5%.

Techniques: Hybridization

Journal: Pulmonary Circulation

Article Title: Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

doi: 10.1086/681272

Figure Lengend Snippet: Prx1 regulates transforming growth factor (TGF)–β signaling in embryonic lungs. A, Immunohistochemical staining for TGF-β1 and TGF-β2 in lung sections derived from E15.5 (top) and D0 (bottom) Prx1WT (left) and Prx1null (right) mice. Brown indicates positive staining of antibodies. While TGF-β localization is restricted to the developing vessel area (arrows) in Prx1WT lungs, in Prx1null lungs TGF-β expression is observed in areas other than the vessel wall vicinity (arrowheads). Hematoxylin staining (blue) is used for nuclear counterstaining. Scale bar = 40 μm. B, Western blotting for TGF-β1 protein expression in E17.5 lungs from Prx1WT and Prx1null mice. C, Real-time polymerase chain reaction (PCR) for TGF-β1 messenger RNA (mRNA) expression in E17.5 lungs from Prx1WT and Prx1null mice. D, Western blotting for phosphorylated Smad2 (P-Smad2; top) and total Smad 2/3 (middle) in E17.5 lungs from Prx1WT and Prx1null mice. E, Real-time PCR for plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in E17.5 lungs from Prx1WT and Prx1null mice. For Western immunoblotting (B, D), β-actin was used as a loading control. For mRNA expression by real-time PCR (C, E), target gene expression was normalized to GAPDH. Two asterisks indicate P < 0.01. WT: wild type.

Article Snippet: The following primary antibodies were used for section staining: anti-fibrillin (Fbn) 1 and Fbn2 rabbit polyclonal antibodies (gift of R. Mecham, University of Washington), anti-tropoelastin polyclonal antibody (Abcam), anti-α-smooth muscle actin (SMA) monoclonal antibody (clone 1A4; Sigma-Aldrich), anti-γ-SMA monoclonal antibody (clone B4; Santa Cruz Biotech), anti-SM22α polyclonal antibody (Abcam), anti–smooth muscle myosin heavy chain (SMMHC) monoclonal antibody (Abcam), anti-TGF-β1 polyclonal antibody (Promega), and anti-TGF-β2 polyclonal antibody (R&D Systems).

Techniques: Immunohistochemical staining, Staining, Derivative Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Control, Targeted Gene Expression

Journal: Pulmonary Circulation

Article Title: Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

doi: 10.1086/681272

Figure Lengend Snippet: 10T1/2 cell differentiation to a smooth muscle phenotype is transforming growth factor (TGF)–β dependent. A, Immunostaining for α–smooth muscle actin (SMA), γ-SMA, SM22α, and smooth muscle myosin heavy chain (SMMHC; all positive reactions are green) in 10T1/2 cells, with or without TGF-β1 treatment, in the presence or absence of a TGF-β type I receptor (TGF-βRI) inhibitor (LY364947). Scale bar = 10 μm. B, α- and γ-SMA messenger RNA expression in 10T1/2 cells after stimulation with TGF-β1, with or without LY364947. Plasminogen activator inhibitor type 1 (PAI-1) was used as a positive control for canonical TGF-β signaling activation. Signals were normalized to GAPDH. C, Western immunoblotting for α- and γ-SMA expression in 10T1/2 cells after stimulation with TGF-β1, with or without a TGF-βRI inhibitor (SB431542). Phosphorylation of Smad2 (P-Smad2) was examined as a marker of canonical TGF-β signaling. GAPDH was used as a loading control.

Article Snippet: The following primary antibodies were used for section staining: anti-fibrillin (Fbn) 1 and Fbn2 rabbit polyclonal antibodies (gift of R. Mecham, University of Washington), anti-tropoelastin polyclonal antibody (Abcam), anti-α-smooth muscle actin (SMA) monoclonal antibody (clone 1A4; Sigma-Aldrich), anti-γ-SMA monoclonal antibody (clone B4; Santa Cruz Biotech), anti-SM22α polyclonal antibody (Abcam), anti–smooth muscle myosin heavy chain (SMMHC) monoclonal antibody (Abcam), anti-TGF-β1 polyclonal antibody (Promega), and anti-TGF-β2 polyclonal antibody (R&D Systems).

Techniques: Cell Differentiation, Immunostaining, RNA Expression, Positive Control, Activation Assay, Western Blot, Expressing, Phospho-proteomics, Marker, Control

Journal: Pulmonary Circulation

Article Title: Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

doi: 10.1086/681272

Figure Lengend Snippet: Transforming growth factor (TGF)–β–dependent 10T1/2 cell differentiation tightly interacts with Prx1-dependent extracellular matrix (ECM) stiffness. A, Schema for the stiffness-controlled polyacrylamide (PAA) gel 2-D culture system. B, 10T1/2 cells were cultured on PAA gel 2-D substrates with a stiffness of either 500 (top) or 1,500 (bottom) Pa, which is parallel to the E17.5 Prx1-lung stiffness, in the presence or absence of TGF-β stimulation. Smooth muscle cell differentiation was evaluated by expression of α–smooth muscle actin (SMA; left, green), SM22α (middle, green), and smooth muscle myosin heavy chain (SMMHC; right, red). 4′,6‐Diamidino‐2‐phenylindole dihydrochloride (blue) was used for nuclear staining. Scale bar = 10 μm. C, Aspect ratio of cells cultured on 2-D PAA gels under the above-described conditions was examined to quantify cell morphology change. D–F, Real-time polymerase chain reaction results for 10T1/2 cells cultured on 2-D PAA gels of either 500- or 1,500-Pa stiffness with or without TGF-β stimulation. α-SMA (D), SM22α (E), and SMMHC (F) messenger RNA expression was examined and normalized by GAPDH or cyclophilin A (cyclo A) expression. One asterisk indicates P < 0.05, and two asterisks indicate P < 0.01. G, 3-D projection of confocal images of F-actin-stained 10T1/2 cells cultured in soft and stiff 3-D methacrylated hyaluronic acid synthetic scaffolds. F-actin is red, nucleus is blue. Scale bar = 20 μm.

Article Snippet: The following primary antibodies were used for section staining: anti-fibrillin (Fbn) 1 and Fbn2 rabbit polyclonal antibodies (gift of R. Mecham, University of Washington), anti-tropoelastin polyclonal antibody (Abcam), anti-α-smooth muscle actin (SMA) monoclonal antibody (clone 1A4; Sigma-Aldrich), anti-γ-SMA monoclonal antibody (clone B4; Santa Cruz Biotech), anti-SM22α polyclonal antibody (Abcam), anti–smooth muscle myosin heavy chain (SMMHC) monoclonal antibody (Abcam), anti-TGF-β1 polyclonal antibody (Promega), and anti-TGF-β2 polyclonal antibody (R&D Systems).

Techniques: Cell Differentiation, Cell Culture, Expressing, Staining, Real-time Polymerase Chain Reaction, RNA Expression

Journal: Biological & pharmaceutical bulletin

Article Title: Microfibril-Associated Protein 5 Contributes to the Elastic Fiber Abnormalities in Aged Skin.

doi: 10.1248/bpb.b24-00828

Figure Lengend Snippet: Fig. 2. MFAP5 Strongly Co-localized with Aggregated Tropoelastin-Positive Fibers

Article Snippet: After 24 h, the conditioned medium and cell lysates were collected (see Supplementary Materials for further details) and subjected to immunoblotting according to previously described methods.9) The antibodies used in this study are as follows: anti-tropoelastin antibody (Supplementary Materials); anti-MFAP5 antibody (Proteintech Japan, Tokyo, Japan); horseradish peroxidase-conjugated antiGAPDH antibody; anti-DYKDDDK (FLAG) tag antibody; anti-HA tag antibody; anti-V5 tag antibody (all from Wako); and anti-His-tag mAb (Clone: OGHis) (MBL, Tokyo, Japan).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Microfibril-Associated Protein 5 Contributes to the Elastic Fiber Abnormalities in Aged Skin.

doi: 10.1248/bpb.b24-00828

Figure Lengend Snippet: Fig. 3. Exogenous Addition of MFAP5 Induces Aggregation of Elastic Fibers

Article Snippet: After 24 h, the conditioned medium and cell lysates were collected (see Supplementary Materials for further details) and subjected to immunoblotting according to previously described methods.9) The antibodies used in this study are as follows: anti-tropoelastin antibody (Supplementary Materials); anti-MFAP5 antibody (Proteintech Japan, Tokyo, Japan); horseradish peroxidase-conjugated antiGAPDH antibody; anti-DYKDDDK (FLAG) tag antibody; anti-HA tag antibody; anti-V5 tag antibody (all from Wako); and anti-His-tag mAb (Clone: OGHis) (MBL, Tokyo, Japan).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Increased Risk of Aortic Dissection with Perlecan Deficiency

doi: 10.3390/ijms23010315

Figure Lengend Snippet: RNA expression of the major components of elastic fibers in Hspg2 −/− -Tg mice without AD. RNA expression of the major components of elastic fibers in Hspg2 −/− -Tg mice at 10 weeks of age was not significantly different from that in WT-Tg mice. In Hspg2 −/− -Tg mice at 20 weeks of age, fibulin-4 ( FBLN4 ) expression was significantly decreased. RNA expression levels were normalized to that of β-actin and are indicated relative to the latter. (Mean ± SEM, n = 5, * p < 0.05, vs. WT-Tg at 20 weeks of age).

Article Snippet: After blocking with PVDF blocking reagent (TOYOBO, Osaka, Japan), the membrane was incubated with anti-tropoelastin monoclonal antibody (1:2000; Elastin Products Company, Inc.), anti-fibrillin-1 polyclonal antibody (1:1000; Santa Cruz Biotechnology) or anti-β-actin monoclonal antibody (1:2000; Santa Cruz Biotechnology) and diluted with blocking reagent overnight.

Techniques: RNA Expression, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Increased Risk of Aortic Dissection with Perlecan Deficiency

doi: 10.3390/ijms23010315

Figure Lengend Snippet: qPCR analysis of SMC-specific markers in the aortic tissue of Hspg2 −/− -Tg mice without AD at 10- and 20-weeks of age. RNA expression of SMC-specific marker genes, Acta2 and Myh11 , was significantly decreased in Hspg2 −/− -Tg mice. Myocardin ( Myocd ) expression in Hspg2 −/− -Tg mice was not significantly different from that in WT-Tg mice. RNA expression levels were normalized to that of β-actin and are indicated relative to the latter. (Mean ± SEM, n = 5, * p < 0.05 vs. WT-Tg).

Article Snippet: After blocking with PVDF blocking reagent (TOYOBO, Osaka, Japan), the membrane was incubated with anti-tropoelastin monoclonal antibody (1:2000; Elastin Products Company, Inc.), anti-fibrillin-1 polyclonal antibody (1:1000; Santa Cruz Biotechnology) or anti-β-actin monoclonal antibody (1:2000; Santa Cruz Biotechnology) and diluted with blocking reagent overnight.

Techniques: RNA Expression, Marker, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Increased Risk of Aortic Dissection with Perlecan Deficiency

doi: 10.3390/ijms23010315

Figure Lengend Snippet: Expression and activity of matrix metalloproteinase (MMPs). ( A ) RNA expression of matrix metalloproteinase in Hspg2 −/− -Tg mice without AD showed no significant difference compared to that in WT-Tg mice. RNA expression levels were normalized to that of β-actin and are indicated as relative to the latter. (Mean ± SEM, 10 w; n = 6, 20 w; n = 3) ( B ) In gelatin zymography, MMP-2 activity did not differ, and MMP-9 activity was not detected in the aorta of WT-Tg and Hspg2 −/− -Tg mice without AD. MMP2 activity is indicated relative to pro-MMP2 activity. (Mean ± SEM, n = 3).

Article Snippet: After blocking with PVDF blocking reagent (TOYOBO, Osaka, Japan), the membrane was incubated with anti-tropoelastin monoclonal antibody (1:2000; Elastin Products Company, Inc.), anti-fibrillin-1 polyclonal antibody (1:1000; Santa Cruz Biotechnology) or anti-β-actin monoclonal antibody (1:2000; Santa Cruz Biotechnology) and diluted with blocking reagent overnight.

Techniques: Expressing, Activity Assay, RNA Expression, Zymography

Journal: International Journal of Molecular Sciences

Article Title: Increased Risk of Aortic Dissection with Perlecan Deficiency

doi: 10.3390/ijms23010315

Figure Lengend Snippet: Maturity of elastic fiber in aortic tissue. ( A ) The amount of desmosine, an indicator of elastic fiber maturity, was evaluated with an amino acid analysis. The amount of desmosine in the aortic tissue of 20-week-old Hspg2 −/− -Tg mice without AD was significantly decreased compared to that in age-matched WT-Tg mice or 10-week-old Hspg2 −/− -Tg mice (Mean ± SEM, n = 5, # p , * p < 0.05 vs. Hspg2 −/− -Tg at 20 weeks). ( B ) Immaturity of elastic fibers was evaluated by western blotting with anti-tropoelastin(TE), soluble precursor elastin, antibody, or anti-fibrillin-1 antibody. Each band was quantified using ImageJ software and is shown relative to β-actin. Soluble tropoelastin in Hspg2 −/− -Tg mouse aortic tissue was significantly increased compared to that in WT-Tg mice (Mean ± SEM, n = 3, # p , * p < 0.05 vs. WT-Tg, respectively).

Article Snippet: After blocking with PVDF blocking reagent (TOYOBO, Osaka, Japan), the membrane was incubated with anti-tropoelastin monoclonal antibody (1:2000; Elastin Products Company, Inc.), anti-fibrillin-1 polyclonal antibody (1:1000; Santa Cruz Biotechnology) or anti-β-actin monoclonal antibody (1:2000; Santa Cruz Biotechnology) and diluted with blocking reagent overnight.

Techniques: Western Blot, Software