Journal: Redox Biology

Article Title: Nogo-B mediates endothelial oxidative stress and inflammation to promote coronary atherosclerosis in pressure-overloaded mouse hearts

doi: 10.1016/j.redox.2023.102944

Figure Lengend Snippet: Nogo-B mediated excess ROS production in HUVECs upon TNF-α stimulation. ( A ) Representative confocal images of MitoSOX Red fluorescence in HUVECs. (B ) Flow cytometric analysis of MitoSOX Red staining-indicated mitochondrial ROS level and quantification by MFI from scrambled (NC) or Nogo-B siRNA under vehicle or TNFα stimuli for 24 h n = 8/group. ( C ) Representative images of DCF fluorescence and ( D ) Intensity statistics and distribution of DCF fluorescence images in HUVECs under 24-h TNFα stimulation. n ≥ 80 (DCF-DA Green) cells from 6 independent experiments. ( E ) representative traces and ( F ) statistics of HPLC analysis of DHE oxidative products in HUVECs lysates transfected with scrambled (NC) or Nogo-B siRNA under vehicle or TNFα stimuli for 24 h. n = 7 independent experiments for each group. ( G ) NOX activity assays in TNFα-stimulated HUVECs transfected with NC or si-Nogo-B. n = 5/group. ( H ) Western blot analysis of NOX2 in TNFα-stimulated HUVECs transfected with NC or si-Nogo-B. n = 5/group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Polyvinylidene fluoride membranes were incubated at 4 °C overnight using the following antibodies: Nogo-A/B (1:2000, #ab47085, Abcam); VCAM-1 (1:2000, #T55427, Abmart); ICAM-1 (1:500, #T40024, Abmart); P38 (1:1000, #9212, CST); p-P38 (1:1000, #9216, CST); P65 (1:1000, #8242, CST); p-P65 (1:1000, #3033, CST); AKT (1:1000, #9272, CST); P-AKT (1:1000, #9271, CST); IP3R1 (1:500, #ACC-019, Alomone); NgBR (1:1000, #TN25175S, Abmart); NOX2 (1:1000, #19013-1-AP, proteintech); NOX1 (1:1000, #17772-1-AP, Proteintech); NOX4 (1:1000, #14347-1-AP, proteintech); OPA1 (1:1000, #67589, CST); Drp1 (1:1000, #5391, CST); Mfn1 (1:1000, #66776-1-Ig, proteintech); Mfn2 (1:1000, #12186-1-AP, proteintech); VDAC1 (1:2000, #66345-1-Ig, proteintech); MCU(1:2000, #26312-1-AP, proteintech); Fis1(1:500, #10956-1-AP, proteintech) and GAPDH (1:5000, #HRP-60004, proteintech), followed by the rabbit or mouse with HRP-conjugated secondary antibody.

Techniques: Fluorescence, Staining, Transfection, Activity Assay, Western Blot

Journal: Redox Biology

Article Title: Nogo-B mediates endothelial oxidative stress and inflammation to promote coronary atherosclerosis in pressure-overloaded mouse hearts

doi: 10.1016/j.redox.2023.102944

Figure Lengend Snippet: Nogo-B-activated ROS-p38-p65 pathway was responsible for adhesion molecules upregulation in ECs. ( A-B ) Reprsentative Western blotting images and quantifications of protein levels of VCAM-1 and Nogo-B in HUVECs transfected with NC or si-Nogo-B upon 24-h TNFα stimulation in the presence or absence of 20 μM Mitotempo ( A, n = 7/group) or 5 mM NAC ( B, n = 5/group). ( C ) Western blot analysis of VCAM-1, NOX2 and Nogo-B in HUVECs under 24-h TNFα stimuli in the presence or absence of 50 μM GSK2795039 (NOX2 inhibitors). n = 7 per group. ( D-E ) Western blot analysis of VCAM-1 and Nogo-B in whole heart tissue after 24-h TAC treated with vehicle or NAC or infliximab (a chimeric anti-TNFα monoclonal antibody). n = 5/group.

Article Snippet: Polyvinylidene fluoride membranes were incubated at 4 °C overnight using the following antibodies: Nogo-A/B (1:2000, #ab47085, Abcam); VCAM-1 (1:2000, #T55427, Abmart); ICAM-1 (1:500, #T40024, Abmart); P38 (1:1000, #9212, CST); p-P38 (1:1000, #9216, CST); P65 (1:1000, #8242, CST); p-P65 (1:1000, #3033, CST); AKT (1:1000, #9272, CST); P-AKT (1:1000, #9271, CST); IP3R1 (1:500, #ACC-019, Alomone); NgBR (1:1000, #TN25175S, Abmart); NOX2 (1:1000, #19013-1-AP, proteintech); NOX1 (1:1000, #17772-1-AP, Proteintech); NOX4 (1:1000, #14347-1-AP, proteintech); OPA1 (1:1000, #67589, CST); Drp1 (1:1000, #5391, CST); Mfn1 (1:1000, #66776-1-Ig, proteintech); Mfn2 (1:1000, #12186-1-AP, proteintech); VDAC1 (1:2000, #66345-1-Ig, proteintech); MCU(1:2000, #26312-1-AP, proteintech); Fis1(1:500, #10956-1-AP, proteintech) and GAPDH (1:5000, #HRP-60004, proteintech), followed by the rabbit or mouse with HRP-conjugated secondary antibody.

Techniques: Western Blot, Transfection

Journal: Frontiers in Immunology

Article Title: Identification of biomarkers related to immune and inflammation in membranous nephropathy: comprehensive bioinformatic analysis and validation

doi: 10.3389/fimmu.2023.1252347

Figure Lengend Snippet: Analysis of immune-associated hub genes. (A) Lollipop plot of CSF1R correlation with 28 immune cells. The horizontal coordinate indicates the correlation size, and the color indicates the P-value. (B) Immune-related GSEA analysis of CSF1R with different expression levels. (C) Distribution of 28 immune cells in CSF1R with different expression levels. (D-F) Analysis of CYBB. **P <0.01, ***P <0.001, ****P <0.0001.

Article Snippet: To measure the protein levels of CSF-1 and CYBB/NOX2, we used specific ELISA kits for human CSF-1 (KE00184; Proteintech, USA) and CYBB/NOX2 (EK13559; Signalway Antibody, USA).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Identification of biomarkers related to immune and inflammation in membranous nephropathy: comprehensive bioinformatic analysis and validation

doi: 10.3389/fimmu.2023.1252347

Figure Lengend Snippet: Validation of CSF-1 and CYBB. Each point represents a sample. (A, B) ELISA measurement of CSF-1 and CYBB. (C, D) mRNA expression of CSF1R and CYBB was analyzed by RT-qPCR. *** P < 0.001, vs Control.

Article Snippet: To measure the protein levels of CSF-1 and CYBB/NOX2, we used specific ELISA kits for human CSF-1 (KE00184; Proteintech, USA) and CYBB/NOX2 (EK13559; Signalway Antibody, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Probucol mitigates high-fat diet-induced cognitive and social impairments through disruption of redox-inflammation association

doi: 10.1101/2023.09.05.556289

Figure Lengend Snippet: ( A-D ) Western blotting analysis of cerebral cortex and hippocampus lysates of three groups mice are shown in A and C . Brain extracts of male mice were analyzed by Western blotting using antibodies against iNOS, NOX2, TNFα and GAPDH. The levels of the proteins in the mice cortex and hippocampus are quantified in B and D , respectively (n = 4 mice per group, ordinary one-way ANOVA, followed by Tukey’s multiple comparisons test). HFD-feeding started 8 weeks before treating the male C57BL/6J mice with 0.005% probucol in drinking water for 12 weeks.

Article Snippet: In this study, the following primary antibodies were used for immunoblotting: iNOS Polyclonal Antibody (Proteintech, 18985-1-AP), NOX2 Polyclonal Antibody (Proteintech, 19013-1-AP), TNF Alpha Polyclonal Antibody (Proteintech, 17590-1-AP), and GAPDH antibody (Proteintech, 60004-1-Ig).

Techniques: Western Blot