anti cd46 258 mem  (Novus Biologicals)

Journal: Journal of dairy science

Article Title: Yak milk-derived exosomes alleviate lipopolysaccharide-induced intestinal inflammation by inhibiting PI3K/AKT/C3 pathway activation.

doi: 10.3168/jds.2021-20175

Figure Lengend Snippet: Figure 3. Protein–protein interaction network of the CD46 proteins and immune-related proteins and tight junction proteins. Fifteen pro- teins were linked into the network. CDH1 = E-cadherin; OCLN = occluding; TJP1 = ZO-1; LTA = TNF-β. Known interactions are represented:

Article Snippet: The membranes were blocked with 5% skim milk-TBS-Tween20 for 4 h at room temperature and incubated overnight at 4°C with the primary antibodies (diluted 1:1,000 in PBS) against proteins of membrane CD46 (cat. # NB500–301, Novus Biologicals Europe; 1:800), HSP-90α (cat. # 8165, Cell Signaling; 1:1,000), CHI3L1 (cat. # LS-B8213, Life Span BioSciences), APOH (cat. # LS-C314171, Life Span BioSciences, 1:300), tight junction protein 1 (ZO1; cat. # 21773–1-AP, Proteintech; 1:1,000), E-cadherin (cat. # 22018–1-AP, Proteintech; 1:1000), AKT (cat. # 10176–2-AP, Proteintech), phosphorylated (P)-AKT (Ser473; cat. # 4060, Cell Signaling), PI3K (cat. # 2524, Cell Signaling), phosphorylated (P)-PI3K (cat. # 4249T, Cell Signaling), NFκ-B (cat. # 10745–1-AP, Proteintech), C3 (cat. # 21337–1-AP, Proteintech), and β-actin (cat. # abs132184, Absin, Absin Bioscience Co. Ltd.; 1:500).

Techniques:

Journal: Scientific Reports

Article Title: Complement regulatory protein CD46 induces autophagy against oxidative stress-mediated apoptosis in normal and asthmatic airway epithelium

doi: 10.1038/s41598-018-31317-5

Figure Lengend Snippet: Increased apoptosis and decreased CD46 in the nasal mucosa of the asthmatic patients. ( A ) CD46 expression (red arrow) was increased in the asthmatic patients (n = 10) compared with the healthy controls (n = 10) as shown by immunohistochemical staining. Epithelial cell shedding and a decreased expression of CD46 (red arrow) were noted in the fragile epithelium of the asthmatic patients (n = 10). Representative confocal microscopic analysis between intact nasal epithelium ( B ) and fragile epithelium ( C ) from the asthmatic patients. Primary nasal epithelium biopsy stained with TUNEL (FITC), CD46 (PE), and 4′,6-diamidino-2-phenylindole (DAPI) (nuclear stain). The yellow arrows indicate TUNEL-positive cells. Decreased CD46 expression and increased TUNEL staining in the fragile upper airway epithelial cells are shown. Scale bar, 20 um. ( D , E ) Statistical data of 10 paired experiments between intact and fragile epithelium from asthmatic patients for the number of CD46 and TUNEL-positive cells (% epithelial cells stained) per high-power field. The Kruskal-Wallis test was used. * p < 0.05.

Article Snippet: Confluent monolayer primary nasal epithelial cells or A549 cells (human bronchial epithelial cell line, American Type Culture Collection, Rockville, MD) were cultured with or without anti-human CD46 mAb (5 ug/ml) (Clone: MEM-258) (GeneTex, San Antonio, Texas, USA) in RPMI-1640 medium containing 10% fetal bovine serum (Gibco).

Techniques: Expressing, Immunohistochemical staining, Staining, TUNEL Assay

Journal: Scientific Reports

Article Title: Complement regulatory protein CD46 induces autophagy against oxidative stress-mediated apoptosis in normal and asthmatic airway epithelium

doi: 10.1038/s41598-018-31317-5

Figure Lengend Snippet: Analysis of CD46 and cleaved CASPASE-3A activity from Der p 2-mediated apoptosis of primary upper airway epithelial cells in the asthmatic patients and control subjects. ( A ) Primary nasal epithelial cells from the asthmatic patients stimulated with Der p 2 (10 μg/ml) and CD46 expression analyzed at 12, 24 and 48 hours by flow cytometry. Representative profiles are shown. ( B ) Western blot analysis was performed to detect CD46 and cleaved CASPASE-3A activity from Der p 2-treated epithelial cells for 48 hours from the asthmatic patients and control subjects. Statistical data of 30 paired experiments between asthmatic patients and control subjects for the expressions of CD46 ( C ) and cleaved CASPASE-3A ( D ) after Der p 2 stimulation for 48 hours. The Kruskal-Wallis test (among multiple groups) and Wilcoxon signed ranked test (after Der p 2) were used. *p < 0.05 after Der p2 and # p < 0.05 compared to the control group.

Article Snippet: Confluent monolayer primary nasal epithelial cells or A549 cells (human bronchial epithelial cell line, American Type Culture Collection, Rockville, MD) were cultured with or without anti-human CD46 mAb (5 ug/ml) (Clone: MEM-258) (GeneTex, San Antonio, Texas, USA) in RPMI-1640 medium containing 10% fetal bovine serum (Gibco).

Techniques: Activity Assay, Control, Expressing, Flow Cytometry, Western Blot

Journal: Scientific Reports

Article Title: Complement regulatory protein CD46 induces autophagy against oxidative stress-mediated apoptosis in normal and asthmatic airway epithelium

doi: 10.1038/s41598-018-31317-5

Figure Lengend Snippet: CD46 induced autophagy in primary upper airway epithelial cells from the controls and asthmatic patients. Cells were incubated for 4 hours in complete medium in the presence of anti-CD46 mAb (5 μg/ml), isotype control antibody (IgG), or in nutrient-deprived media (starvation) and/or the autophagy inhibitor 3-methyladenine (3-MA) (10 mmol/L) (Sigma-Aldrich, St. Louis, MO). ( A ) Representative images of GFP-LC3 puncta (autophagosomes) in nasal epithelial cells are shown by confocal microscopy. ( B ) The cytosolic soluble form of LC3-I was converted into the autophagic vesicle-associated form of LC3-II and was used as a marker of autophagosome formation. The number of GFP-LC3 vesicles per cell in primary nasal epithelial cells was calculated from 200 cells for each experiment. Quantification of GFP-LC3 puncta per cell was assayed and the statistical data are shown. ( C ) Immunoblotting was used to analyze the LC3- II expression in primary upper airway epithelial cells from the controls and asthmatic patients. ( D ) Statistical data of the experiments with 30 paired samples as shown. ( E ) Primary nasal epithelial cells from the asthmatic subjects were treated with bafilomycin, and immunoblotting was used to analyze the expressions of LC3-II. ( F ) Statistical data of the experiments with six paired samples as shown. The Kruskal-Wallis test was used to determine significant differences. * p < 0.05.

Article Snippet: Confluent monolayer primary nasal epithelial cells or A549 cells (human bronchial epithelial cell line, American Type Culture Collection, Rockville, MD) were cultured with or without anti-human CD46 mAb (5 ug/ml) (Clone: MEM-258) (GeneTex, San Antonio, Texas, USA) in RPMI-1640 medium containing 10% fetal bovine serum (Gibco).

Techniques: Incubation, Control, Confocal Microscopy, Marker, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Complement regulatory protein CD46 induces autophagy against oxidative stress-mediated apoptosis in normal and asthmatic airway epithelium

doi: 10.1038/s41598-018-31317-5

Figure Lengend Snippet: CD46 induced autophagy against H2O2-induced airway epithelial cell apoptosis in the controls and asthmatic patients. ( A ) To detect the role of CD46-induced autophagy in early apoptotic cells with Annexin V-positive but PI-negative primary nasal epithelium cells after exposure to hydrogen peroxide, a FITC Annexin V/propidium iodide Apoptosis Detection Kit I (BD Pharmingen, USA) was used. CD46 mAb (5 μg/ml) and/or autophagy inhibitor 3-methyladenine (3-MA) (10 mmol/L) (Sigma-Aldrich, St. Louis, MO) was co-incubated with primary nasal epithelium cells (1 × 10 5 cells) from the asthmatic patients and then cultured with H2O2 (0.5 mM) for 1 hour followed by 8 hours recovery. Representative figures are shown. ( B ) Statistical data of the experiments with 30 paired samples. The Kruskal-Wallis test was used to determine significant differences. * p < 0.05 after treatment and # p < 0.05 compared to the control group.

Article Snippet: Confluent monolayer primary nasal epithelial cells or A549 cells (human bronchial epithelial cell line, American Type Culture Collection, Rockville, MD) were cultured with or without anti-human CD46 mAb (5 ug/ml) (Clone: MEM-258) (GeneTex, San Antonio, Texas, USA) in RPMI-1640 medium containing 10% fetal bovine serum (Gibco).

Techniques: Incubation, Cell Culture, Control

Journal: Scientific Reports

Article Title: Complement regulatory protein CD46 induces autophagy against oxidative stress-mediated apoptosis in normal and asthmatic airway epithelium

doi: 10.1038/s41598-018-31317-5

Figure Lengend Snippet: CD46 inhibited IL-1β and IL-6 from H2O2-activated airway epithelial cells from the asthmatic patients. Supernatant from anti-CD46 mAb (5 μg/ml)-pretreated primary nasal epithelium cells from the asthmatic patients after exposure to H2O2 (0.5 mM) was analyzed using 8-OHdG ( A ) and IL-1β ( B ) and IL-6 ( C ) ELISA-based systems. The Kruskal-Wallis test was used. Statistical data of the experiments with 30 paired samples are shown. *p < 0.05.

Article Snippet: Confluent monolayer primary nasal epithelial cells or A549 cells (human bronchial epithelial cell line, American Type Culture Collection, Rockville, MD) were cultured with or without anti-human CD46 mAb (5 ug/ml) (Clone: MEM-258) (GeneTex, San Antonio, Texas, USA) in RPMI-1640 medium containing 10% fetal bovine serum (Gibco).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Complement regulatory protein CD46 induces autophagy against oxidative stress-mediated apoptosis in normal and asthmatic airway epithelium

doi: 10.1038/s41598-018-31317-5

Figure Lengend Snippet: Silencing ATG5 decreased CD46-activated autophagy in A549 cells. A549 cells were transfected with small interfering RNAs (siRNA) targeting human ATG5 or control siRNA. The A549 cells were incubated for 4 hours in complete medium in the presence of anti-CD46 mAb (5 μg/ml), isotype control antibody (IgG), or in nutrient-deprived media (starvation). Proteins were extracted from the A549 cells and analyzed by Western blot using anti-ATG5 mAb, anti-LCB3-1 mAb, anti- anti-LCB3-2 mAb. The Kruskal-Wallis test was used to determine significant differences. Statistical data of the experiments are shown. *p < 0.05 after siRNA and # p < 0.05 compared to the IgG group.

Article Snippet: Confluent monolayer primary nasal epithelial cells or A549 cells (human bronchial epithelial cell line, American Type Culture Collection, Rockville, MD) were cultured with or without anti-human CD46 mAb (5 ug/ml) (Clone: MEM-258) (GeneTex, San Antonio, Texas, USA) in RPMI-1640 medium containing 10% fetal bovine serum (Gibco).

Techniques: Transfection, Control, Incubation, Western Blot

Journal: Scientific Reports

Article Title: Complement regulatory protein CD46 induces autophagy against oxidative stress-mediated apoptosis in normal and asthmatic airway epithelium

doi: 10.1038/s41598-018-31317-5

Figure Lengend Snippet: CD46 enhanced the expression of GOPC and inhibited the expressions of PRO-IL-1β and NLRP3 in H2O2-activated A549 cells. ( A ) A549 cells were incubated for 4 hours in complete medium in the presence of anti-CD46 mAb (5 μg/ml), or isotype control antibody (IgG). Immunoblotting of anti-GOPC mAb was used to analyze the GOPC expression. Statistical data of experiments on six paired samples are shown. * p < 0.05 ( B ) The anti-CD46 mAb (5 μg/ml)-pretreated A549 cells after exposure to H2O2 (0.2 mM) and cultured with the autophagy inhibitor 3-methyladenine (3-MA) (10 mmol/L) were analyzed by Western blot using anti-IL-1β mAb and anti-NLRP3 mAb. The Kruskal-Wallis test was used to determine significant differences. Statistical data of experiments with six paired samples are shown. *p < 0.05.

Article Snippet: Confluent monolayer primary nasal epithelial cells or A549 cells (human bronchial epithelial cell line, American Type Culture Collection, Rockville, MD) were cultured with or without anti-human CD46 mAb (5 ug/ml) (Clone: MEM-258) (GeneTex, San Antonio, Texas, USA) in RPMI-1640 medium containing 10% fetal bovine serum (Gibco).

Techniques: Expressing, Incubation, Control, Western Blot, Cell Culture