Journal: American Journal of Physiology - Cell Physiology

Article Title: Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function

doi: 10.1152/ajpcell.00151.2020

Figure Lengend Snippet: Protein expression of the copper transporter ATP7A is decreased in blood vessels from caveolin-1 knockout (KO) mice (Cav-1−/−). A: relative protein expression of ATP7A and Atox1 in aortas from Cav-1 wild-type (WT) and Cav-1−/− mice was determined by Western blotting with antibodies specific to respective proteins. Densitometric analysis is shown (right, n = 5). B: ATP7A mRNA expression in aortas from Cav-1 WT and Cav-1−/− mice was determined by real-time quantitative RT-PCR (n = 3). C: protein expression for ATP7A and Atox1 in mouse fibroblasts isolated from Cav-1 WT and Cav-1−/− mice (n = 3). D: Cav-1 WT and Cav-1−/− mouse fibroblasts mice were transfected with Cav-1 WT plasmid. Lysates were used to measure ATP7A, Cav-1, and actin protein expression. (n = 3). E: superoxide dismutase (SOD3)-specific activity in conditional medium of Cav-1 WT plasmid transfected Cav-1−/− mouse fibroblast cells. Cav-1 WT and Cav-1−/− mouse fibroblast cells were transfected with Cav-1-WT plasmid. The specific activity of SOD3 was determined by the ratio of activity to relative amount of protein in culture conditional medium. Results are presented as means ± SE. *P < 0.05, NS, not significant. ATP7a, Menkes ATPase, copper-transporting P-type ATPase.

Article Snippet: The following primary antibodies were used: anti-Atox1 (homemade) ( 23 ), anti-SOD3 (homemade) ( 13 ), anti-SOD1 (ab16831; Abcam), anti-ATP7A (B8162; LifeSpan Biosciences), anti-actin (SC-1616; Santa Cruz Biotechnology), anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Cav-1 (610407 or 610060; BD Transduction), anti-paxillin (612405; BD Transduction), anti-flotillin-1 (610820; BD Transduction), anti-heparan sulfate (MAB2040; Millipore), anti-fibulin-5 (12188; Proteintech) or anti-collagen type-1 (Proteintech, 14695).

Techniques: Expressing, Knock-Out, Western Blot, Quantitative RT-PCR, Isolation, Transfection, Plasmid Preparation, Activity Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function

doi: 10.1152/ajpcell.00151.2020

Figure Lengend Snippet: Caveolin-1 (Cav-1) is required for ATP7A protein stabilization and prevents proteosomal degradation. A: Cav-1 wild-type (WT) and Cav-1−/− [knockout (KO)] mouse fibroblasts were lysed and immunoprecipitated (IP) with anti-ATP7A, followed by immunoblotting (IB) with an anti-ubiquitin antibody. Right: averaged data for ATP7A ubiquitination (n = 3). B: mouse fibroblast cells were incubated with an inhibitor of the proteasome MG132 (20 μmol/L) for 24 h or an inhibitor of the lysosome chloroquine (100 μmol/L) for 24 h, and the protein expression of ATP7A was determined by Western blot (n = 3). C: membrane fractionation of mouse aorta. Equivolume fractions isolated from the top (fraction 1) to the bottom (fraction 13) were immunoblotted with antibodies as indicated. D: IP of Cav-1 using an anti-Cav-1 antibody in aortic lysates, followed by IB with an ATP7A antibody (n = 3). Results are presented as means ± SE. *P < 0.05. ATP7a, Menkes ATPase, copper-transporting P-type ATPase.

Article Snippet: The following primary antibodies were used: anti-Atox1 (homemade) ( 23 ), anti-SOD3 (homemade) ( 13 ), anti-SOD1 (ab16831; Abcam), anti-ATP7A (B8162; LifeSpan Biosciences), anti-actin (SC-1616; Santa Cruz Biotechnology), anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Cav-1 (610407 or 610060; BD Transduction), anti-paxillin (612405; BD Transduction), anti-flotillin-1 (610820; BD Transduction), anti-heparan sulfate (MAB2040; Millipore), anti-fibulin-5 (12188; Proteintech) or anti-collagen type-1 (Proteintech, 14695).

Techniques: Knock-Out, Immunoprecipitation, Western Blot, Incubation, Expressing, Fractionation, Isolation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function

doi: 10.1152/ajpcell.00151.2020

Figure Lengend Snippet: Transgenic mice overexpressing Menkes ATPase, copper-transporting P-type ATPase (ATP7A; ATP7A Tg) rescued superoxide dismutase (SOD3) activity and endothelium-dependent relaxation in caveolin-1 knockout (KO) (Cav-1−/−) mice. A: protein levels of ATP7A, SOD3, SOD1, Cav-1, and actin in aortas from Cav-1 wild-type (WT) or Cav-1−/− double Tg mice overexpressing ATP7A were measured (n = 3). B: specific activity of SOD3 and SOD1 in tissue homogenates were assayed as described in Fig. 1. C: endothelium-dependent or -independent relaxation of mesenteric resistance arteries from Cav-1 WT or Cav-1−/− double Tg mice overexpressing ATP7A. Vasorelaxation was evoked by acetylcholine (ACh) and sodium nitroprusside (SNP) after preconstriction with phenylephrine (n = 6). D: membrane fractionation of mouse aorta of Cav-1 WT, Cav-1−/−, and ATP7A-Tg/Cav-1−/− mice. Equal amounts of caveolin-enriched lipid raft fractions (caveolae/lipid rafts; fraction 4 to 5) and non-caveolae/lipid rafts; fractions 10 to 13) from a pool of 4 mouse aortas were immunoblotted with antibodies as indicated. Results are presented as means ± SE. *P < 0.05. ATP7a, Menkes ATPase, copper-transporting P-type ATPase.

Article Snippet: The following primary antibodies were used: anti-Atox1 (homemade) ( 23 ), anti-SOD3 (homemade) ( 13 ), anti-SOD1 (ab16831; Abcam), anti-ATP7A (B8162; LifeSpan Biosciences), anti-actin (SC-1616; Santa Cruz Biotechnology), anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Cav-1 (610407 or 610060; BD Transduction), anti-paxillin (612405; BD Transduction), anti-flotillin-1 (610820; BD Transduction), anti-heparan sulfate (MAB2040; Millipore), anti-fibulin-5 (12188; Proteintech) or anti-collagen type-1 (Proteintech, 14695).

Techniques: Transgenic Assay, Activity Assay, Knock-Out, Fractionation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function

doi: 10.1152/ajpcell.00151.2020

Figure Lengend Snippet: Proposed working model showing how caveolin-1 (Cav-1) is required for stabilizing protein expression of the copper transporter ATP7A and enabling copper delivery to extracellular superoxide dismutase 3 (SOD3) in vascular tissue to enable its full activity and protection of endothelial function against oxidative stress. ATP7A, Menkes ATPase, copper (Cu)-transporting P-type ATPase; Ub, ubiquitin.

Article Snippet: The following primary antibodies were used: anti-Atox1 (homemade) ( 23 ), anti-SOD3 (homemade) ( 13 ), anti-SOD1 (ab16831; Abcam), anti-ATP7A (B8162; LifeSpan Biosciences), anti-actin (SC-1616; Santa Cruz Biotechnology), anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Cav-1 (610407 or 610060; BD Transduction), anti-paxillin (612405; BD Transduction), anti-flotillin-1 (610820; BD Transduction), anti-heparan sulfate (MAB2040; Millipore), anti-fibulin-5 (12188; Proteintech) or anti-collagen type-1 (Proteintech, 14695).

Techniques: Expressing, Activity Assay

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: (A) The mRNA and protein expression levels of FLJ10540 were determined by Q-RT-PCR and western blotting in fibulin-5 transfectants. The result of mRNA was normalized against the expression level of GAPDH mRNA in each fibulin-5-stable clones. For protein analyses, the cell lysates (50 μg) of Hone1/fibulin-5 transfectants was subjected to immunoblot analysis with anti-FLJ10540 and β-actin antibodies. β-actin was used as a control. (B) Fibulin-5 siRNAs decreased the expression levels of FLJ10540 mRNA and protein in fibulin-5-NPC transfectants. The Q-RT-PCR and western blotting were performed as in (B). (C) The luciferase assays were done to detect promoter activities of FLJ10540 in cotransfected with in a dose-dependent manner of DDK-, DDK-fibulin-5-, negative control and sifibulin-5 in Hone1 cells. The luciferase activity in 1μg cell lysate was normalized to β-galactosidase activity. Data are representative of three independent experiments done in triplicates. The Western blotting of DDK-fibulin-5 in a dose-dependent manner was shown in the right side of the left panel. (D) ChIP analysis of endogenous FLJ10540 promoter in the presence and absence of fibulin-5 in Hone 1 cells. The protein-DNA complexes were immunoprecipitated with DDK and IgG antibodies, and FLJ10540 promoter element was detected by PCR. (E) Migration and invasion decreased in cells transfected with FLJ10540 siRNA in vehicle-Hone1 and fibulin-5-Hone1 transfectants. The relative-fold migration and invasion values for the stable clones were normalized against the vehicle cells and are represented diagrammatically. All data represent the mean ± SD of 3 independent experiments.

Article Snippet: After antigen retrieval, the sections were incubated with diluted anti-FLJ10540 antibody (polyclonal; generated by us; 1:500; polyclonal; Abnova, Taiwan 1:100) and anti-fibulin-5 (polyclonal; Abnova, Taiwan 1:100; monoclonal; Protein Tech Group, Inc, Chicago, USA; 1:50) at room temperature for 1 hour, followed by washing with PBS.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Clone Assay, Control, Luciferase, Negative Control, Activity Assay, Immunoprecipitation, Migration, Transfection

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: The correlation between fibulin-5 and FLJ10540 expression in NPC.

Article Snippet: After antigen retrieval, the sections were incubated with diluted anti-FLJ10540 antibody (polyclonal; generated by us; 1:500; polyclonal; Abnova, Taiwan 1:100) and anti-fibulin-5 (polyclonal; Abnova, Taiwan 1:100; monoclonal; Protein Tech Group, Inc, Chicago, USA; 1:50) at room temperature for 1 hour, followed by washing with PBS.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: (A) Hone1-expressing FLJ10540 cells were serum-starved for 24 hour and treated with or without AKT inhibitor. The Total cell lysates of vehicle-Hone and FLJ10540-Hone1, transfected cells were immunoblotted for the unphosphorylated and phosphorylated forms of AKT. β-actin was used as the internal loading control. (B) A negative control and FLJ10540 siRNAs were transfected into Hone1 cells for 24 hour and western blotting was performed as in (B). (C) Vehicle-Hone1 and FLJ10540-Hone1 transfected cells were serum-starved and treated with the AKT inhibitor for 24 hour. The migration and invasion ratios of vehicle-Hone1 and fibulin-5-Hone1 transfected cells were determined as previously described.

Article Snippet: After antigen retrieval, the sections were incubated with diluted anti-FLJ10540 antibody (polyclonal; generated by us; 1:500; polyclonal; Abnova, Taiwan 1:100) and anti-fibulin-5 (polyclonal; Abnova, Taiwan 1:100; monoclonal; Protein Tech Group, Inc, Chicago, USA; 1:50) at room temperature for 1 hour, followed by washing with PBS.

Techniques: Expressing, Transfection, Control, Negative Control, Western Blot, Migration