rabbit polyclonal antibody against cav 1  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: SMAD-Independent Down-Regulation of Caveolin-1 by TGF-β: Effects on Proliferation and Survival of Myofibroblasts

doi: 10.1371/journal.pone.0116995

Figure Lengend Snippet: A and B , Cultured IMR-90 cells were treated with or without TGF-β1 (2 ng/ml) for 24 h and total RNA was isolated. A, cDNA Superarray analysis was performed and mRNA expression levels of Cav-1 and cyclophilin A are shown. Densitometric ratios of Cav-1:cyclophilin A are also shown. B, Histogram of real-time RT-PCR for Cav-1 expression after treated with TGF-β1 (2 ng/ml for 24 h) using triplicate samples from at least three individual experiments, normalized to 18S as mean±S.E.M. * p <0.05 compared to control. C , IMR-90 cells were treated with TGF-β1 (0–5 ng/ml) for 24 h and cell lysates extracted. Cell lysates were subjected to SDS-PAGE and immunoblotted with an antibody against Cav-1; the blot was stripped and probed for β-tubulin. Densitometric ratios of Cav-1:β-tubulin are also shown on the right, as mean±S.E.M. D , IMR-90 cells were stimulated with TGF-β1 (2 ng/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and immunoblotted with an antibody against Cav-1; the blot was then stripped and probed for β-tubulin. Densitometric ratios of Cav-1:β-tubulin are shown on the right, as mean±S.E.M. Results are averages of at least three independent experiments.

Article Snippet: Rabbit polyclonal antibody (N-20) against a peptide at the amino terminus of human Cav-1 was purchased from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Cell Culture, Isolation, Expressing, Quantitative RT-PCR, SDS Page

Journal: PLoS ONE

Article Title: SMAD-Independent Down-Regulation of Caveolin-1 by TGF-β: Effects on Proliferation and Survival of Myofibroblasts

doi: 10.1371/journal.pone.0116995

Figure Lengend Snippet: A , IMR-90 cells were treated with inhibitors of p38 MAPK (SB203580; 6 μM) or ALK5 (SB431542; 0.5 μM) for 30 min prior to treatment with or without TGF-β1 (2 ng/ml) for a period of 48 h. Cell lysates were extracted and Western immunoblotting performed with an antibody against Cav-1; the blot was then stripped and probed for β-tubulin. B , Densitometric analyses of blots in (A) showed as % inhibition of baseline Cav-1 protein expression levels treated with TGF-β1. *indicates effect of SB203580 to completely block the inhibitory effect of TGF-β1 on Cav-1 expression. Results are averages of at least three independent experiments. Data are presented as mean±S.E.M. C , IMR-90 cells stably transfected with a kinase-deficient p38 MAPK (pcDNA-p38KM) or control vector (pcDNA) were treated with or without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were obtained and subjected to SDS-PAGE and immunoblotted for Cav-1 and α-smooth muscle actin (α-SMA); blots were stripped and probed for β-tubulin. D , IMR-90 cells stably expressing SMAD2 sh RNA (pSU6H- sh SMAD2) or control vector (pSU6H) were treated with/without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were immunoblotted for SMAD2, Cav-1 and α-SMA. The blots were stripped and probed for β-tubulin.

Article Snippet: Rabbit polyclonal antibody (N-20) against a peptide at the amino terminus of human Cav-1 was purchased from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Western Blot, Inhibition, Expressing, Blocking Assay, Stable Transfection, Transfection, Plasmid Preparation, SDS Page

Journal: PLoS ONE

Article Title: SMAD-Independent Down-Regulation of Caveolin-1 by TGF-β: Effects on Proliferation and Survival of Myofibroblasts

doi: 10.1371/journal.pone.0116995

Figure Lengend Snippet: A , Human lung fibroblasts (IMR-90) were stably transfected with a plasmid encoding Cav-1 (pRC/CMV2-Cav1) or with empty vector (pRC/CMV2). Localization of Cav-1 protein was then analyzed by immunofluoresence staining with a rabbit polyclonal antibody to Cav-1. B , Stably-transfected cells described in (A) were treated with/without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were obtained and subjected to immunoblotting for Cav-1, α-smooth muscle actin (α-SMA), and β-tubulin. C , Stably-transfected cells described in (A) were serum-deprived for 24 h and then treated with/without TGF-β1 (2 ng/ml) for 48 h followed by stimulation with 10% fetal bovine serum for 24 h. Cell numbers were assessed both prior to and after serum stimulation with an automated Coulter counter (n = 6 per group) as mean±S.E.M. *indicates p < 0.05 vs. control pRC/CMV2 “fibroblasts”. **indicates p < 0.05 vs. pRC/CMV2 “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. D , Cells in (C) were labeled with BrdU during the 24 h of serum stimulation (n = 6 per group). BrdU assays were performed as described in “Methods”. *indicates p < 0.05 vs. control pRC/CMV2 “fibroblasts”. **indicates p < 0.05 vs. pRC/CMV2 “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. E , Quiescent stably transfected IMR-90 cells described in (A) were treated with/without TGF-β1 (2 ng/ml) for 5 days in serum-free medium. Apoptosis assays using an ELISA for ss DNA (n = 6 for each group) were performed as described in “Methods”. *indicates p < 0.05 vs. control pRC/CMV2 “fibroblasts”. **indicates p < 0.01 vs. pRC/CMV2 “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments.

Article Snippet: Rabbit polyclonal antibody (N-20) against a peptide at the amino terminus of human Cav-1 was purchased from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Staining, Western Blot, Labeling, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: SMAD-Independent Down-Regulation of Caveolin-1 by TGF-β: Effects on Proliferation and Survival of Myofibroblasts

doi: 10.1371/journal.pone.0116995

Figure Lengend Snippet: A , IMR-90 cells stably transfected with a plasmid encoding sh RNA targeted against Cav-1 (pSU6H- sh Cav1) or with control plasmid (pSU6H) were treated with/without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were obtained and Western blots for Cav-1, α-smooth muscle actin (α-SMA) and β-tubulin performed. B , Stably-transfected cells described in (A) were serum-deprived for 24 h and treated with/without TGF-β1 (2 ng/ml) for 48 h followed by stimulation with 10% FBS for 24 h. Cell counts were assessed both prior to and after serum stimulation with an automated Coulter counter (n = 6 per group) shown as mean±S.E.M. *indicates p < 0.05 vs. control pSU6H “fibroblasts”. **indicates p < 0.05 vs. pSU6H “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. C , Stably transfected cells described in (A) were serum-deprived for 24 h and treated with/without TGF-β1 (2 ng/ml) for 48 h followed by BrdU labeling for 24 h in the presence of 10% FBS (n = 6 per group) shown as mean±S.E.M. *indicates p < 0.05 vs. control pSU6H “fibroblasts”. Similar results were obtained from 3 independent experiments. D , Quiescent stably-transfected cells described in ( A ) were treated with/without TGF-β1 (2 ng/ml) for 5 days and apoptotic assay for ss DNA performed as described in “Methods” (n = 6 per group) shown as mean±S.E.M. *indicates p < 0.05 vs. control pSU6H “fibroblasts”. **indicates p < 0.05 vs. pSU6H “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments.

Article Snippet: Rabbit polyclonal antibody (N-20) against a peptide at the amino terminus of human Cav-1 was purchased from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Labeling

Journal: PLoS ONE

Article Title: SMAD-Independent Down-Regulation of Caveolin-1 by TGF-β: Effects on Proliferation and Survival of Myofibroblasts

doi: 10.1371/journal.pone.0116995

Figure Lengend Snippet: TGF-β1 activates the cell surface TGF-β receptor(s) complex that leads to rapid activation of the canonical SMAD pathway as well as the SMAD- in dependent p38 MAPK pathway. Activation of the SMAD pathway is required for the induction of a cellular program of growth-arrest and myofibroblast differentiation. In contrast, activation of the p38 MAPK pathway, independently of SMAD2/3, is required the down-regulation of Cav-1 by TGF-β1. Down-regulation of Cav-1 by TGF-β1 “primes” differentiated myofibroblasts for enhanced proliferative responses to mitogens and resistance to apoptosis. These divergent TGF-β signaling pathways may explain, in part, the contextual effects of TGF-β1 as both a growth-inhibitor and –promoter on the same target (mesenchymal) cells.

Article Snippet: Rabbit polyclonal antibody (N-20) against a peptide at the amino terminus of human Cav-1 was purchased from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Activation Assay

Journal: Oncotarget

Article Title: Characterization of physiochemical properties of caveolin-1 from normal and prion-infected human brains

doi: 10.18632/oncotarget.19431

Figure Lengend Snippet: A . Western blotting of caveolin-1 from ten areas were examined including frontal cortex (Fr), parietal cortex (Par), temporal cortex (Te), occipital cortex (Oc), cerebellum (Ce), thalamus (Th), substantial nigra (Sn), putamen (Put), globus pallidus (Gp), and hippocampus (Hip). B . Western blotting of caveolin-1 between non-CJD and CJD patients. β-actin was used as to monitor amount of samples loaded in each lane. C . Western blotting of caveolin-1 of normal human brain homogenate from non-linear sucrose gradient sedimentation fractions. All Western blots are representative of three independent experiments.

Article Snippet: Anti-PrP antibody 3F4 (PrP107-112) [ ], Rabbit anti-caveolin-1 polyclonal antibody Cav-1 (pAb) against caveolin-1 residues 1-97 (BD Biosciences), rabbit monoclonal antibody Cav-1 (N-20) against caveolin-1 residues1-20 (Epitomics), and C-term against caveolin-1 residues 167-178 (antibodies-online Inc, Atlanta, GA) were used.

Techniques: Western Blot, Sedimentation

Journal: Oncotarget

Article Title: Characterization of physiochemical properties of caveolin-1 from normal and prion-infected human brains

doi: 10.18632/oncotarget.19431

Figure Lengend Snippet: A . Western blotting of caveolin-1 in both detergent-soluble and- insoluble fractions probed with anti-caveolin-1 antibody. B . Western blotting of total caveolin-1 (S1), detergent-soluble (S2) and detergent-insoluble (P2) fractions from size exclusion chromatography (FPLC) probed with anti-caveolin-1 antibody. C . Chromatogram of size exclusion chromatography of human brain homogenate from a non-CJD patient. D . 2D electrophoresis analysis of caveolin-1 from insoluble fraction. The caveolin-1 recovered in soluble fraction is shown in the insert box. The above Western blots are representative of three independent experiments.

Article Snippet: Anti-PrP antibody 3F4 (PrP107-112) [ ], Rabbit anti-caveolin-1 polyclonal antibody Cav-1 (pAb) against caveolin-1 residues 1-97 (BD Biosciences), rabbit monoclonal antibody Cav-1 (N-20) against caveolin-1 residues1-20 (Epitomics), and C-term against caveolin-1 residues 167-178 (antibodies-online Inc, Atlanta, GA) were used.

Techniques: Western Blot, Size-exclusion Chromatography, Two-Dimensional Gel Electrophoresis

Journal: Oncotarget

Article Title: Characterization of physiochemical properties of caveolin-1 from normal and prion-infected human brains

doi: 10.18632/oncotarget.19431

Figure Lengend Snippet: A . Western blotting of brain homogenates from the frontal cortex of CJD and non-CJD patients treated with PK at different concentrations ranging from 0 to 2,000 μg/ml probing with anti-caveolin-1 antibody. B . Brain homogenate from a sCJD patient treated with different amounts of PK probing with anti-caveolin-1 or anti-PrP antibody 3F4. The above Western blots are representative of three independent experiments.

Article Snippet: Anti-PrP antibody 3F4 (PrP107-112) [ ], Rabbit anti-caveolin-1 polyclonal antibody Cav-1 (pAb) against caveolin-1 residues 1-97 (BD Biosciences), rabbit monoclonal antibody Cav-1 (N-20) against caveolin-1 residues1-20 (Epitomics), and C-term against caveolin-1 residues 167-178 (antibodies-online Inc, Atlanta, GA) were used.

Techniques: Western Blot

Journal: Oncotarget

Article Title: Characterization of physiochemical properties of caveolin-1 from normal and prion-infected human brains

doi: 10.18632/oncotarget.19431

Figure Lengend Snippet: A . Western blotting of detergent-soluble (S2) and detergent-insoluble (P2) caveolin-1 molecules after PK-treatment in sCJD and non-CJD cases probing with anti-caveolin-1 antibody. The untreated caveolin-1 migrated between 23 and 24 kDa and PK-treated one migrated between 19 and 20 kDa. B . The α- and β-isoforms of caveolin-1 from both S2 and P2 fractions migrated between 22 and 24 kDa without PK treatment, two bands migrating between ˜19 and 21 KDa treated with PK at 5 μg/ml, only one band migrating at ˜19-20 KDa was detectable in the samples treated with PK at 25 μg/ml or higher. The above Western blots are representative of three independent experiments.

Article Snippet: Anti-PrP antibody 3F4 (PrP107-112) [ ], Rabbit anti-caveolin-1 polyclonal antibody Cav-1 (pAb) against caveolin-1 residues 1-97 (BD Biosciences), rabbit monoclonal antibody Cav-1 (N-20) against caveolin-1 residues1-20 (Epitomics), and C-term against caveolin-1 residues 167-178 (antibodies-online Inc, Atlanta, GA) were used.

Techniques: Western Blot

Journal: Oncotarget

Article Title: Characterization of physiochemical properties of caveolin-1 from normal and prion-infected human brains

doi: 10.18632/oncotarget.19431

Figure Lengend Snippet: Both pAb (Cav-1) against residues1-97 and Cav-1 (N-20) against residues 1-20 detected caveolin-1 in A . A431 cell line and B . human brain tissues. Arrows represent α- and β-isoforms of caveolin-1, respectively, in A . Arrow heads represent full-length or truncated forms in B . The above Western blots are representative of three independent experiments.

Article Snippet: Anti-PrP antibody 3F4 (PrP107-112) [ ], Rabbit anti-caveolin-1 polyclonal antibody Cav-1 (pAb) against caveolin-1 residues 1-97 (BD Biosciences), rabbit monoclonal antibody Cav-1 (N-20) against caveolin-1 residues1-20 (Epitomics), and C-term against caveolin-1 residues 167-178 (antibodies-online Inc, Atlanta, GA) were used.

Techniques: Western Blot

Journal: Oncotarget

Article Title: Characterization of physiochemical properties of caveolin-1 from normal and prion-infected human brains

doi: 10.18632/oncotarget.19431

Figure Lengend Snippet: Caveolin-1 from cultured A431 cells or human brain tissues was treated with or without PK prior to Western blotting probed with three different antibodies against caveolin-1. pAb (Cav-1) against residues1-97 and Cav-1 (N-20) against residues 1-20 detected caveolin-1 while antibody C-term that against C-term region of caveolin only detected caveolin-1 from the cultured cells but not from the brains. The above Western blots are representative of three independent experiments.

Article Snippet: Anti-PrP antibody 3F4 (PrP107-112) [ ], Rabbit anti-caveolin-1 polyclonal antibody Cav-1 (pAb) against caveolin-1 residues 1-97 (BD Biosciences), rabbit monoclonal antibody Cav-1 (N-20) against caveolin-1 residues1-20 (Epitomics), and C-term against caveolin-1 residues 167-178 (antibodies-online Inc, Atlanta, GA) were used.

Techniques: Cell Culture, Western Blot