rabbit polyclonal antibody against human gapdh  (Novus Biologicals)

Journal: bioRxiv

Article Title: Enhanced Omicron subvariant cross-neutralization efficacy of a monovalent SARS-CoV-2 BA.4/5 mRNA vaccine encoding a noncleaved, nonfusogenic spike antigen

doi: 10.1101/2023.09.10.557088

Figure Lengend Snippet: a , Schematic of the challenge experiment schedule. Eight-week-old female BALB/c mice were immunized as described in ( n = 5 for each group and n = 6 for UnVac). Two weeks after the booster dose, blood was collected via tail clip bleeding on day 28. Immunized mice were challenged intranasally with 1 × 10 5 PFU of SARS-CoV-2 BA.5 on day 33. Mice were sacrificed 2 days post-infection to measure the tissue viral load in the lungs. b , Levels of binding IgG against Omicron BA.5 RBD in sera collected two weeks post booster immunization. c , d , Tissue viral load measured by plaque assay ( c ) and RT-qPCR ( d ) in the lung homogenate (tops of boxes indicate mean values). Each data point represents the viral load from two technical duplicates for RT-qPCR and two biological duplicates for plaque assay. In b,c, Non-detected (ND) antibody and viral load levels are assigned values half of the LOD for graphical representation. e–g , Cytokine levels in the lung homogenates were measured by ELISA. Each data point represents the cytokine concentration from two technical replicates. Samples with concentrations below the assay LOD were assigned a value of zero. h , Immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining of the lung sections. Lung sections for IHC were stained with a SARS-CoV-2 N-specific monoclonal antibody (tailed arrow) and CD3-specific polyclonal antibody (arrowheads). Scale bar, 100 μm. Statistical significance was determined by a Student t -test on the log 10 -transformed data. ns, not significant. The dotted lines indicate the LOD.

Article Snippet: Antibodies were obtained as follows: mouse monoclonal antibody targeting the C-terminal region of the SARS-CoV-2 S protein (GeneTex, Irvine, CA, USA; cat. no. GTX632604); rabbit polyclonal antibody targeting the N-terminal region of the SARS-CoV-2 S protein (GeneTex; cat. no. GTX135384); mouse monoclonal antibody against SARS-CoV nucleocapsid (N) protein (Sino Biological; cat. no. 40143-MM08); rabbit polyclonal antibody targeting N-terminus region of human ACE2 (Genetex; cat. no. GTX101395); rabbit polyclonal antibody against human GAPDH (Abcam, Cambridge, UK; cat. no. ab9485); mouse monoclonal antibody targeting the 872–891 amino acid sequence within the human α-actinin (Santa Cruz Biotechnology Inc, Dallas, TX, USA; cat. no. sc-166524); mouse monoclonal antibody against MLV p30 (Abcam; cat. no. ab130757).

Techniques: Infection, Binding Assay, Plaque Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay, Immunohistochemistry, Staining, Transformation Assay

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

doi: 10.3390/ijms23031259

Figure Lengend Snippet: Pathogen-specific regulation of a disintegrin and metalloproteinase (ADAM)10 protein expression and surface localization in bacterial infection. A549 cells were grown to confluence and either left unstimulated or infected with Pseudomonas aeruginosa (P. aeruginosa ) (multiplicity of infection of 5 (MOI 5) ( A , B ), infected with Streptococcus pneumoniae ( S. pneumoniae ) (MOI 5) ( C , D ) or stimulated with exotoxin A (ExoA) (100 ng/mL, E , F ). In ( A – E ), samples were taken after an incubation time of 30, 60, 120 or 240 min. In ( F ), samples were probed after 4 h. ( A , C , E ): ADAM10 protein expression and maturation was investigated in cell lysates by Western blot probing with an antibody against the C-terminus (intracellular part). Probing against glyceraldehyde-3-phosphat dehydrogenase (GAPDH) served as loading control. Band intensities of the pro-form (100 kDa) and the mature form (70 kDa) were quantified by densitometry and normalized to the expression of the unstimulated cells (0 h). A representative blot is shown in ( A , right) (antibody specificity detailed in ). ( B , D , E ): ADAM10 surface expression was investigated by surface staining with an N-terminal antibody against ADAM10 (1 µg/mL) and an APC-coupled secondary antibody (5 µg/mL) and subsequent flow cytometric analysis (quantification as mean fluorescence intensity). The values of the adequate isotype control were subtracted followed by normalization to the unstimulated cells. A representative histogram is shown in ( B , left). Quantitative data are shown as means + SD of three independent experiments. Asterisks indicate significance difference to the control calculated using two tailed two samples t-test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Mouse monoclonal antibody against human CD9 (MM2/57); Rabbit polyclonal antibody against human ADAM10 C-terminus was obtained from Invitrogen (Frankfurt, Germany), rabbit polyclonal antibody against human GAPDH from Santa Cruz Biotech (Dallas, TX, USA).

Techniques: Expressing, Infection, Incubation, Western Blot, Control, Staining, Fluorescence, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

doi: 10.3390/ijms23031259

Figure Lengend Snippet: Pathogen-specific activation of ADAM10 depends on the toxin repertoire and calcium increase. ( A ) A549 cells were grown to confluence and either left unstimulated or infected with heat-inactivated P. aeruginosa (multiplicity of infection of 5 (MOI 5). Samples were taken after an incubation time of 30, 60, 120 or 240 min. ADAM10 protein expression and maturation was investigated in cell lysates by Western blot probing with an antibody against the C-terminus (intracellular part). Probing against GAPDH served as loading control. Band intensities of the pro-form (100 kDa) and the mature form (70 kDa) were quantified by densitometry and normalized to the expression of the unstimulated cells (0 h). A representative blot is shown in ( A ). ( B , C , D ) A549 cells were grown to 70% confluency on poly-L-lysine coated glass coverslips and either left unstimulated (PBS) or stimulated with ExoA (100 ng/mL) for 4 h in Tyrode’s solution ( B ), calcium free Tyrode’s solution ( C , D ). Subsequently, the cells loaded with 5 μM Fura-2 AM for 45 min at 37 °C followed by calcium signaling recording over 1000 s in Tyrode’s solution ( B ), calcium free Tyrode’s solution ( C ) or calcium free Tyrode’s solution followed by addition of 2 mM calcium after 120 s of calcium signaling recording ( D ). Quantitative data are shown as means + SD of three independent experiments. Asterisks indicate significance difference among treated cells at the indicated time point calculated using two-way ANOVA and Bonferroni post-test for F340/380 ratio over time and one-way ANOVA and Tukey post-test for area under the curve (* p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001, n.s. not significant). In ( A ), significance was analyzed by two tailed two samples t-test. No significant differences were observed.

Article Snippet: Mouse monoclonal antibody against human CD9 (MM2/57); Rabbit polyclonal antibody against human ADAM10 C-terminus was obtained from Invitrogen (Frankfurt, Germany), rabbit polyclonal antibody against human GAPDH from Santa Cruz Biotech (Dallas, TX, USA).

Techniques: Activation Assay, Infection, Incubation, Expressing, Western Blot, Control, Two Tailed Test

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: The association of neuron-derived orphan receptor 1 with pulmonary vascular remodeling in COPD patients

doi: 10.2147/copd.s151820

Figure Lengend Snippet: Figure 1 Hypoxemia induced NOR1 upregulation and pulmonary vascular remodeling. Notes: Lung tissues were collected and pulmonary vascular remodeling was estimated. (A) HE staining showed the wall thickness of pulmonary vessels in patients. (B) Immunohistochemistry staining (α-SMA) showed the muscularized vessels of pulmonary vessels in patients. (C and D) Histogram showed the results of wall thickness and muscularized vessel percentage in patients. (E and F) Protein levels (SDS-PAGE and histogram) of HIF-1α, α-SMA, NOR1, and cyclin D1 in peripheral lung tissues of different patients. *P,0.01 as compared with control. Control: n=15; hypoxemia: n=11. Abbreviations: NOR1, neuron-derived orphan receptor 1; HE, hematoxylin and eosin; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Article Snippet: Patients and methods reagents Rabbit polyclonal antibody against GAPDH (sc-367714) human cyclin D1 (sc-718) or smooth muscle myosin heavy chain 11 (ab53219) and mouse monocloal antibody against smooth muscle actin (sc-53142) or BrdU (sc-32323) were all purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

Techniques: Staining, Immunohistochemistry, SDS Page, Control, Derivative Assay, Polyacrylamide Gel Electrophoresis

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: The association of neuron-derived orphan receptor 1 with pulmonary vascular remodeling in COPD patients

doi: 10.2147/copd.s151820

Figure Lengend Snippet: Figure 5 NOR1 promoted proliferation via cyclin D1. Notes: PASMCs were cultured in hypoxia (5% O2) or normoxia (21% O2) after different treatments. (A and B) Cyclin D1 protein levels (SDS-PAGE and histogram) in PASMCs after transfection with NOR1 overexpression plasmid (pNOR1). (C and D) Cyclin D1 protein levels (SDS-PAGE and histogram) in PASMCs after transfection with NOR1-siRNA or NC-siRNA. (E) BrdU incorporation in hypoxic or normoxic cells after PD0332991 (a specific inhibitor of cyclin D1-CDK4/6 complex) treatment. (F) Histogram shows the results of BrdU incorporation. (G) Results of cell counts in hypoxic or normoxic cells after PD0332991 treatment. #P,0.05 as compared with control (or normoxia), *P,0.05 as compared with NC-siRNA, &P,0.05 as compared with DMSO (n=6). Abbreviations: NC, negative control; NOR1, neuron-derived orphan receptor 1; PASMC, pulmonary arterial smooth muscle cell; SDS-PAGE, sodium dodecyl sulfate– polyacrylamide gel electrophoresis.

Article Snippet: Patients and methods reagents Rabbit polyclonal antibody against GAPDH (sc-367714) human cyclin D1 (sc-718) or smooth muscle myosin heavy chain 11 (ab53219) and mouse monocloal antibody against smooth muscle actin (sc-53142) or BrdU (sc-32323) were all purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

Techniques: Cell Culture, SDS Page, Transfection, Over Expression, Plasmid Preparation, BrdU Incorporation Assay, Control, Negative Control, Derivative Assay, Polyacrylamide Gel Electrophoresis