goat polyclonal antibody against βgag domain  (R&D Systems)

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Western blots confirm increased levels of versican in the lungs of patients with pulmonary arterial hypertension (PAH). Proteoglycan extracts from lung tissue homogenates treated with the chondroitinase ABC were used. For each sample, 1.5 mg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, and immunoblotting for versican was performed using two different antibodies, mAb 2B1 for the C-terminal G3 domain (A) and pAb 3054 for the βGAG domain (B). Samples from 4 patients with idiopathic PAH (IPAH) were compared with age-matched unused donor lungs (control). Increased versican fragments (lanes 5, 6, and 8) were observed in subjects with IPAH compared with age-matched control subjects. C, Schematic drawing of versican splice variants and recognition sites for mAb 2B1 and pAb 3054. Versican has two globular domains, the G1 domain at the amino terminus and the G3 domain at the carboxy terminus. The splice variants vary in the GAG-attachment domains (αGAG and βGAG). C: complement regulatory region; E: epidermal growth factor–like domain; HABR: hyaluronan-binding region; Ig: immunoglobulin-like domain; L: lectin-binding domain.

Article Snippet: Two different primary antibodies were used to detect versican, a goat polyclonal antibody against βGAG domain (1∶3000, AF 3054, R&D systems) and a mouse monoclonal antibody against C-terminal G3 domain (2B1, 1∶3000, Seikagaku).

Techniques: Western Blot, Polyacrylamide Gel Electrophoresis, Control, Binding Assay

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Versican accumulates in the vascular lesions of individuals with pulmonary arterial hypertension (PAH). A, Immunohistochemistry for versican (in brown) in different vascular lesions. B, Representative images of versican staining in lungs from patients with PAH of different etiologies. A monoclonal antiversican antibody (clone 2B1) against the C-terminal G3 domain was used for immunostaining. Images were captured with a ×20 objective. One section per subject was analyzed (n = 7 for idiopathic PAH [IPAH], n = 7 for failed donor lung as control, n = 3 for atrial septal defect with PAH [ASD], and n = 3 for scleroderma with PAH [Scl]). Donor: healthy donor lungs unused for transplantation as controls; Neg: mouse immunoglobulin G used as negative control.

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Immunohistochemistry, Staining, Immunostaining, Control, Transplantation Assay, Negative Control

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Western blots confirm increased levels of versican in the lungs of patients with pulmonary arterial hypertension (PAH). Proteoglycan extracts from lung tissue homogenates treated with the chondroitinase ABC were used. For each sample, 1.5 mg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, and immunoblotting for versican was performed using two different antibodies, mAb 2B1 for the C-terminal G3 domain (A) and pAb 3054 for the βGAG domain (B). Samples from 4 patients with idiopathic PAH (IPAH) were compared with age-matched unused donor lungs (control). Increased versican fragments (lanes 5, 6, and 8) were observed in subjects with IPAH compared with age-matched control subjects. C, Schematic drawing of versican splice variants and recognition sites for mAb 2B1 and pAb 3054. Versican has two globular domains, the G1 domain at the amino terminus and the G3 domain at the carboxy terminus. The splice variants vary in the GAG-attachment domains (αGAG and βGAG). C: complement regulatory region; E: epidermal growth factor–like domain; HABR: hyaluronan-binding region; Ig: immunoglobulin-like domain; L: lectin-binding domain.

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Western Blot, Polyacrylamide Gel Electrophoresis, Control, Binding Assay

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Versican and inflammation in pulmonary arterial hypertension (PAH). Representative images of double immunostaining for versican (in brown) and CD45 (leukocyte common antigen, in red). A and B, Pronounced perivascular inflammation with little versican staining. C, Infiltration of inflammatory cells in the adventitia without colocalization with versican. D–F, Versican and CD45 are rarely colocalized in plexiform lesions. G and H, Versican accumulates in the neointima, but there is almost no colocalization with inflammatory cells. I–K, Some degree of colocalization in neointima, but it is not consistent. Arrows indicate the colocalization of versican and CD45. The letter L indicates the lumen of pulmonary vessels. Mouse immunoglobulin G (IgG) was used as a negative control. The images were captured with a ×4 objective for K and a ×20 objective for A–J. There were 4–6 sections per slide, and 1 slide per subject was analyzed (n = 13 for PAH group; n = 7 for control group).

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Double Immunostaining, Staining, Negative Control, Control

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Versican and smooth-muscle α-actin–positive cells. Representative images of double immunofluorescence staining for smooth-muscle α-actin (in green) and versican (in red). Nuclear staining was shown in blue. A, Smooth-muscle α-actin staining of neointimal lesion (idiopathic pulmonary arterial hypertension [IPAH]). B, Strong versican immunostaining in the same neointima. C, Merged image (A and B), which shows versican accumulation in areas with smooth-muscle α-actin–positive cells within the neointima. D, Smooth-muscle α-actin staining of plexiform lesion (IPAH). E, Prominent versican staining in the same plexiform lesion. F, Merged image (D and E). G and H, Double immunofluorescence staining of vessels from healthy control subjects, with very low levels of versican in areas with smooth-muscle α-actin–positive cells. I, Rabbit immunoglobulin G–negative control. The images were captured with a ×20 objective. One section per subject was analyzed (n = 4 for IPAH; n = 3 for healthy control subjects).

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Double Immunofluorescence Staining, Staining, Immunostaining, Control, Negative Control

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Upregulation of versican messenger RNA and protein by mechanical strain in human aortic smooth-muscle cells (SMCs) but not in human pulmonary artery smooth-muscle cells (hPASMCs). Results of quantitative polymerase chain reaction (qPCR) for versican isoforms (V0, V1, V2, and V3; A) and a representative Western blot (B) for versican from cell cultures of stretched aortic SMCs and nonstretched controls. Samples were collected after 6 hours of cyclic stretch. Results of qPCR (C) for versican isoforms and a representative Western blot (D) for versican from cell cultures of stretched hPASMCs and nonstretched controls. Samples were collected for another 6 hours after 6 hours of cyclic stretch. For qPCR (A and C), fold changes were calculated using ∆∆Ct method compared with nonstretched controls. β2-microglobulin was used as reference gene (n = 3; asterisk indicates P < 0.05 by Student t test). For Western blots (B and D), equal amount of total proteins from conditioned medium and cell lysates, respectively, were applied to ion-exchange chromatography for proteoglycan purification. Chondroitinase ABC-treated samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing condition and probed with goat antihuman versican β glycosaminoglycan domain antibody for versican isoforms V0 and V1. N: nonstretched control group; S: stretched group; (+): positive control extracted from human aortic SMC–conditioned medium.

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Ion Exchange Chromatography, Purification, Polyacrylamide Gel Electrophoresis, Control, Positive Control

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Versican is regulated by hypoxia. A, quantitative polymerase chain reaction (qPCR) for versican isoforms (V0, V1, V2, and V3) from cell lysates of hypoxic human pulmonary artery smooth-muscle cells (hPASMCs). Data were analyzed using ∆∆Ct method compared with normoxic controls. β2-microglobulin was used as reference gene (n = 3; asterisk indicates P < 0.05 by Student t test). B, Representative Western blot for versican from cell cultures of hypoxic hPASMCs and normoxic controls. Purified, chondroitinase-treated samples were probed with goat antihuman versican β glycosaminoglycan domain antibody for versican isoforms V0 and V1. H: hypoxia; N: normoxia; (+): positive control extracted from human aortic smooth-muscle cell–conditioned medium.

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Purification, Positive Control