recombinant jagged1 protein  (MedChemExpress)

Journal: Journal of Cell Communication and Signaling

Article Title: Unveiling the role of Jagged2 in hypoxic pulmonary arterial hypertension: A NOX2‐mediated pathway

doi: 10.1002/ccs3.70032

Figure Lengend Snippet: Machine learning identification of key genes. (A) Venn diagram of differentially expressed genes (DEGs) and weighted gene co‐expression network (WGCNA) module genes; (B) least absolute shrinkage and selection operator regression (LASSO) coefficient selection plot; (C) random forest (RF) identification of key factors, with mean decrease accuracy (left) indicating the importance of genes to model prediction accuracy and mean decrease Gini (right) indicating the importance of genes to model classification purity; (D) Venn diagram of LASSO and RF analysis; (E) box plot of Jag2 and Pcdh19 expression in transcriptome datasets; (F) receiver operating characteristic (ROC) curve of Jag2 in transcriptome datasets, with Y ‐axis representing sensitivity and X ‐axis representing specificity. Normoxia: n = 7; Hypoxia: n = 7.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4°C with primary antibodies diluted in tris‐buffered saline with tween 20 (TBST): Jag2 (bs‐4244R, Bioss), NOX2 (19013‐1‐AP, Proteintech), nuclear factor erythroid 2‐related factor 2 (Nrf2) (16396‐1‐AP, Proteintech), proliferating cell nuclear antigen (PCNA) (10205‐2‐AP, Proteintech), survivin (10508‐1‐AP, Proteintech), SOD2 (24127‐1‐AP, Proteintech), cleaved caspase‐3 (68773‐1‐lg, Proteintech), B‐cell lymphoma 2 (68103‐1‐lg, Proteintech), Bcl‐2‐associated X protein (BAX) (50599‐2‐lg, Proteintech), α‐SMA (14395‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), CD31 (28083‐1‐AP, Proteintech), VE‐cadherin (A25003, Abclonal), and β‐actin (81115‐1‐RR, Proteintech).

Techniques: Expressing, Selection

Journal: Journal of Cell Communication and Signaling

Article Title: Unveiling the role of Jagged2 in hypoxic pulmonary arterial hypertension: A NOX2‐mediated pathway

doi: 10.1002/ccs3.70032

Figure Lengend Snippet: Effects of NADPH oxidase 2 (NOX2) overexpression on proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) under hypoxic conditions. (A) Expression of Jag2 and NOX2 in PASMCs at various time points under hypoxic treatment detected by immunofluorescence. (B) mRNA levels of Jag2 and NOX2 in PASMCs at various time points under hypoxic treatment detected by qRT‐PCR. (C) Cell proliferation levels were measured by the CCK‐8 assay. (D) Cell proliferation levels assessed by EdU staining, scale bar = 50 μm. (E) Protein expression of Jag2, NOX2, proliferating cell nuclear antigen (PCNA), and survivin in different groups was detected by the western blot. (F) Cell migration levels were assessed using a Transwell assay. (G) Protein expression of nuclear factor erythroid 2–related factor 2 (Nrf2) and SOD2 in different groups detected by the western blot; levels of superoxide dismutase (SOD), malondialdehyde (MDA), total glutathione (GSH), reduced GSH, and oxidized GSH measured by assay kits; reactive oxygen species (ROS) levels assessed by 2',7'‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining. (H) Protein expression of caspase‐3, cleaved caspase‐3, B‐cell lymphoma 2 (Bcl2), and Bcl‐2‐associated X protein (BAX) in different groups detected by the western blot. (I) Apoptosis levels in cells of different groups were assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, scale bar = 50 μm. (J) Apoptosis levels in cells of different groups were measured by flow cytometry (FCM). * indicates p < 0.05 compared to the hypoxia + siJag2 group, ** indicates p < 0.01 compared to the hypoxia + siJag2 group; all cell experiments were performed in triplicate.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4°C with primary antibodies diluted in tris‐buffered saline with tween 20 (TBST): Jag2 (bs‐4244R, Bioss), NOX2 (19013‐1‐AP, Proteintech), nuclear factor erythroid 2‐related factor 2 (Nrf2) (16396‐1‐AP, Proteintech), proliferating cell nuclear antigen (PCNA) (10205‐2‐AP, Proteintech), survivin (10508‐1‐AP, Proteintech), SOD2 (24127‐1‐AP, Proteintech), cleaved caspase‐3 (68773‐1‐lg, Proteintech), B‐cell lymphoma 2 (68103‐1‐lg, Proteintech), Bcl‐2‐associated X protein (BAX) (50599‐2‐lg, Proteintech), α‐SMA (14395‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), CD31 (28083‐1‐AP, Proteintech), VE‐cadherin (A25003, Abclonal), and β‐actin (81115‐1‐RR, Proteintech).

Techniques: Over Expression, Migration, Expressing, Immunofluorescence, Quantitative RT-PCR, CCK-8 Assay, Staining, Western Blot, Transwell Assay, Measured Assay, TUNEL Assay, Flow Cytometry

Journal: Journal of Cell Communication and Signaling

Article Title: Unveiling the role of Jagged2 in hypoxic pulmonary arterial hypertension: A NOX2‐mediated pathway

doi: 10.1002/ccs3.70032

Figure Lengend Snippet: Mechanistic study of Jag2 promoting pulmonary artery smooth muscle cell (PASMC) proliferation and migration under hypoxic conditions through the NADPH oxidase 2 (NOX2)/reactive oxygen species (ROS) pathway. (A) CCK‐8 assay to measure cell proliferation levels in each group; (B, C) EdU staining to measure cell proliferation levels in each group (B), with panel C showing the bar graph statistical results of panel B, scale bar = 50 μm; (D) western blot analysis of Jag2, NOX2, proliferating cell nuclear antigen (PCNA), and survivin protein expression in each group; (E) Transwell assay to measure migration levels in each group; (F) western blot analysis of nuclear factor erythroid 2–related factor 2 (Nrf2) and SOD2 protein expression in each group; (G) kit assays to measure superoxide dismutase (SOD) and malondialdehyde (MDA) levels in each group; (H) kit assays to measure total glutathione (GSH), reduced GSH, and oxidized GSH levels in each group; (I) 2',7'‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining to measure ROS levels in each group; (J) western blot analysis of caspase‐3, cleaved caspase‐3, B‐cell lymphoma 2 (Bcl2), and Bcl‐2‐associated X protein (BAX) expression in each group; (K) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to measure apoptosis levels in each group, scale bar = 50 μm; (L) flow cytometry (FCM) to measure apoptosis levels in each group. * p < 0.05 compared to the normoxia group, ** p < 0.01 compared to the normoxia group, # p < 0.05 compared to the hypoxia + siNC group, ## p < 0.01 compared to the hypoxia + siNC group. All experiments were repeated three times.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4°C with primary antibodies diluted in tris‐buffered saline with tween 20 (TBST): Jag2 (bs‐4244R, Bioss), NOX2 (19013‐1‐AP, Proteintech), nuclear factor erythroid 2‐related factor 2 (Nrf2) (16396‐1‐AP, Proteintech), proliferating cell nuclear antigen (PCNA) (10205‐2‐AP, Proteintech), survivin (10508‐1‐AP, Proteintech), SOD2 (24127‐1‐AP, Proteintech), cleaved caspase‐3 (68773‐1‐lg, Proteintech), B‐cell lymphoma 2 (68103‐1‐lg, Proteintech), Bcl‐2‐associated X protein (BAX) (50599‐2‐lg, Proteintech), α‐SMA (14395‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), CD31 (28083‐1‐AP, Proteintech), VE‐cadherin (A25003, Abclonal), and β‐actin (81115‐1‐RR, Proteintech).

Techniques: Migration, CCK-8 Assay, Staining, Western Blot, Expressing, Transwell Assay, TUNEL Assay, Flow Cytometry

Journal: Journal of Cell Communication and Signaling

Article Title: Unveiling the role of Jagged2 in hypoxic pulmonary arterial hypertension: A NOX2‐mediated pathway

doi: 10.1002/ccs3.70032

Figure Lengend Snippet: Regulation of gene expression and vascular remodeling by the Jag2/NADPH oxidase 2 (NOX2) pathway in hypoxic pulmonary arterial hypertension (PAH) rat models. (A) Measurement of mean pulmonary arterial pressure (mPAP), right ventricular systolic pressure (RVSP), pulmonary artery systolic pressure (PASP), and right ventricle (RV)/(left ventricle [LV] + S) values in each group of rats. (B) H&E and Elastica Van Gieson (EVG) staining of pulmonary artery remodeling in each group of rats (scale bar = 50 μm). (C) Transmission electron microscopy of the basal membrane morphology of pulmonary arterioles in each group of rats (scale bar = 5 μm). (D) Western blot analysis of Jag2, NOX2, alpha‐smooth muscle actin (α‐SMA), vimentin, CD31, and VE‐cadherin expression in each group. (E) Immunofluorescence detection of CD31 and α‐SMA expression in the pulmonary arteries of each group of rats (scale bar = 50 μm). (F) Western blot analysis of proliferating cell nuclear antigen (PCNA) and survivin expression in lung tissues of each group. *indicates p < 0.05 compared to the control group; ** indicates p < 0.01 compared to the control group; # indicates p < 0.05 compared to the model + AAV‐shNC group; ## indicates p < 0.01 compared to the model + AAV‐shNC group. N = 8.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4°C with primary antibodies diluted in tris‐buffered saline with tween 20 (TBST): Jag2 (bs‐4244R, Bioss), NOX2 (19013‐1‐AP, Proteintech), nuclear factor erythroid 2‐related factor 2 (Nrf2) (16396‐1‐AP, Proteintech), proliferating cell nuclear antigen (PCNA) (10205‐2‐AP, Proteintech), survivin (10508‐1‐AP, Proteintech), SOD2 (24127‐1‐AP, Proteintech), cleaved caspase‐3 (68773‐1‐lg, Proteintech), B‐cell lymphoma 2 (68103‐1‐lg, Proteintech), Bcl‐2‐associated X protein (BAX) (50599‐2‐lg, Proteintech), α‐SMA (14395‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), CD31 (28083‐1‐AP, Proteintech), VE‐cadherin (A25003, Abclonal), and β‐actin (81115‐1‐RR, Proteintech).

Techniques: Gene Expression, Staining, Transmission Assay, Electron Microscopy, Membrane, Western Blot, Expressing, Immunofluorescence, Control

Journal: Journal of Cell Communication and Signaling

Article Title: Unveiling the role of Jagged2 in hypoxic pulmonary arterial hypertension: A NOX2‐mediated pathway

doi: 10.1002/ccs3.70032

Figure Lengend Snippet: Effects of the Jag2/NADPH oxidase 2 (NOX2) pathway on inflammation, oxidative stress, and apoptosis in a hypoxic pulmonary arterial hypertension (PAH) rat model. (A) Immunohistochemistry detection of CD68 expression in the lungs of different groups of rats, scale bar = 50 μm; (B) ELISA detection of tumor necrosis factor‐alpha (TNF‐α) and IL‐6 levels in serum and lung tissue; (C) measurement of superoxide dismutase (SOD) and malondialdehyde (MDA) levels in the serum of different rat groups; (D) western blot detection of nuclear factor erythroid 2–related factor 2 (Nrf2) and SOD2 expression in lung tissue; (E) DHE staining for reactive oxygen species (ROS) levels in lung tissue, scale bar = 50 μm; (F) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) detection of apoptosis levels in lung tissue, scale bar = 20 μm. * p < 0.05 compared to the control group, ** p < 0.01 compared to the control group, # p < 0.05 compared to the model + AAV‐shNC group, ## p < 0.01 compared to the model + AAV‐shNC group, N = 8.

Article Snippet: The membrane was blocked with 5% nonfat dry milk at room temperature for 1 h. The PVDF membrane was incubated overnight at 4°C with primary antibodies diluted in tris‐buffered saline with tween 20 (TBST): Jag2 (bs‐4244R, Bioss), NOX2 (19013‐1‐AP, Proteintech), nuclear factor erythroid 2‐related factor 2 (Nrf2) (16396‐1‐AP, Proteintech), proliferating cell nuclear antigen (PCNA) (10205‐2‐AP, Proteintech), survivin (10508‐1‐AP, Proteintech), SOD2 (24127‐1‐AP, Proteintech), cleaved caspase‐3 (68773‐1‐lg, Proteintech), B‐cell lymphoma 2 (68103‐1‐lg, Proteintech), Bcl‐2‐associated X protein (BAX) (50599‐2‐lg, Proteintech), α‐SMA (14395‐1‐AP, Proteintech), vimentin (10366‐1‐AP, Proteintech), CD31 (28083‐1‐AP, Proteintech), VE‐cadherin (A25003, Abclonal), and β‐actin (81115‐1‐RR, Proteintech).

Techniques: Immunohistochemistry, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, TUNEL Assay, Control

Journal: Development (Cambridge, England)

Article Title: Nelfb promotes dermal white adipose tissue formation through RNA polymerase II-mediated adipogenic gene regulation

doi: 10.1242/dev.204976

Figure Lengend Snippet: Nelfb is necessary for adipocyte differentiation by promoting the expression of adipocyte transcription factors. (A) Oil Red O staining of control (CTL: Nelfb fl/wt Pdgfra-Cre + or Nelfb wt/wt Pdgfra-Cre + ) and Nelfb −/− ( Nelfb fl/fl Pdgfra-Cre + ) cells cultured in adipocyte differentiation medium for 14 days ( N =4). (B) Staining of CTL and Nelfb −/− cells with antibodies against perilipin 1 (Plin1; mature adipocyte marker: green) and Hoechst 33342 (nuclei stain: blue) ( N =4). (C) RNA-seq analysis of CTL and Nelfb −/− cells cultured in adipocyte differentiation medium for 14 days. Volcano plot shows 762 genes increased (red) and 358 genes decreased (blue) on a log 2 scale upon Nelfb depletion. N =3. (D) Gene Ontology (GO) terms of the 358 decreased and 762 increased genes using Enrichr. (E) Heat map of the 43 genes (≥2-fold change) found in the top five GO terms of decreased genes in D. (F) CUT&RUN on CTL and Nelfb −/− cells cultured in adipocyte differentiation medium for 14 days using an anti-Nelfb and anti-IgG antibody. qPCR analysis was conducted on transcription start site (TSS) regions of Pparg , Cebpa , Cebpb , Stat3 and Krox20. Signal was calculated as a percent of total input DNA ( N =3). (G,H) CUT&RUN using an anti-RNA Polymerase II (RNA Pol II) antibody and anti-IgG antibody ( N =3). qPCR analysis was performed on the TSS (G) or gene body (H) regions of Pparg , Cebpa , Stat3 and Krox20 . (I,J) CUT&RUN using an anti-RNA Polymerase II Ser2 phosphorylation (Pol II Ser2 Phospho) antibody and anti-IgG antibody ( N =3). qPCR analysis was performed on the TSS (I) or gene body (J) regions of Pparg , Cebpa , Stat3 and Krox20 . (K) CUT&RUN using active chromatin mark anti-H3K4me3 and anti-IgG antibody. qPCR analysis on TSS regions of Pparg , Cebpa , Stat3 and Krox20 . Signal was calculated as a percent of total input DNA ( N =3). Data are mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparison tests). ns, not significant. Scale bars: 150 μm.

Article Snippet: Quantities used were: Nelfb: 5 μl/reaction (Cell Signaling Technology, #14894S); RNA Pol II: 3 μl/reaction (Active Motif, #39097); RNA Pol II Ser2 Phosphorylation: 2 μl/reaction (Cell Signaling Technology, #13499S); Nelfe: 5 μl/reaction (Proteintech, #10705-1-AP); IgG: 5 μl/reaction (Cell Signaling Technology, #66362); H3K4me3: 5 μl/reaction (Cell Signaling Technology, #9751S); H3K27me3: 2 μl/reaction (Cell Signaling Technology, #9733S); H3K9me3: 2 μl/reaction (Cell Signaling Technology, #13969S); H3K27Ac: 1 μl/reaction (Cell Signaling Technology, #8173S).

Techniques: Expressing, Staining, Control, Cell Culture, Marker, RNA Sequencing, Phospho-proteomics, Comparison