cytoplasmic protein extraction kit  (Novus Biologicals)

Journal: Cellular and Molecular Neurobiology

Article Title: Nitric Oxide Donor Alleviates Cardiac Arrest Induced Blood Brain Barrier Injury by Inhibiting HMGB1-ATG5 Mediated Endothelial Autophagy

doi: 10.1007/s10571-026-01706-w

Figure Lengend Snippet: JS-K regulates the nucleocytoplasmic translocation of HMGB1 in cerebral microvascular endothelial cells after CA/CPR and OGD/R. A Representative immunoblot images and B quantitation of nuclear HMGB1 and cytoplasmic HMGB1 expression in cerebral microvessels of mice from Sham, CA/CPR and CA/CPR + JS-K groups at 24 h after ROSC. Histone H3 and β-actin were used as the protein loading control. The images of western blotting data derived from triplicate blots conducted as three independent experiments. C Representative immunoblot images and D quantitation of nuclear HMGB1 and cytoplasmic HMGB1 expression in bEnd.3 cells from Control, OGD/R and OGD/R + JS-K groups. Histone H3 and β-actin were used as the protein loading control. The images of western blotting data derived from triplicate blots conducted as three independent experiments. E Representative immunofluorescence images of HMGB1 localization in bEnd.3 cells from Control, OGD/R and OGD/R + JS-K groups. HMGB1 (green), nuclei stained with DAPI (blue). Scale bar = 50 μm. F Quantification of nuclear and cytoplasmic HMGB1 signals. Nuclear HMGB1 was defined as HMGB1 signal overlapping with DAPI, and cytoplasmic HMGB1 as non-overlapping HMGB1 signal

Article Snippet: Cytoplasmic and nuclear protein fractions were isolated from bEnd.3 cells and freshly isolated cerebral microvessels using the nuclear and cytoplasmic protein extraction Kit (WLA020a, Wanleibio, China).

Techniques: Translocation Assay, Western Blot, Quantitation Assay, Expressing, Control, Derivative Assay, Immunofluorescence, Staining

Journal: Cellular and Molecular Neurobiology

Article Title: Nitric Oxide Donor Alleviates Cardiac Arrest Induced Blood Brain Barrier Injury by Inhibiting HMGB1-ATG5 Mediated Endothelial Autophagy

doi: 10.1007/s10571-026-01706-w

Figure Lengend Snippet: Inhibition of HMGB1 nucleocytoplasmic translocation attenuates autophagy and improves endothelial function in bEnd.3 cells. A Representative immunoblot images and B quantitation of nuclear HMGB1, cytoplasmic HMGB1, LC3B II, LAMP2, occludin and ZO-1 expression in bEnd.3 cells from Control, OGD/R and OGD/R + Leptomycin B groups. Histone H3 and β-actin were used as the protein loading control. The images of western blotting data derived from triplicate blots conducted as three independent experiments. C Representative immunofluorescence images of HMGB1 localization in bEnd.3 cells from Control, OGD/R and OGD/R + Leptomycin B groups. HMGB1 (green), nuclei stained with DAPI (blue). Scale bar = 50 μm. D Quantification of nuclear and cytoplasmic HMGB1 signals. Nuclear HMGB1 was defined as HMGB1 signal overlapping with DAPI, and cytoplasmic HMGB1 as non-overlapping HMGB1 signal. E Representative images of fluorescent LC3 dots are shown. F Mean number of autophagosomes and autolysosomes per cell. Scale bar = 10 μm. G The diffusion rates of FITC-dextran (40 kDa). H Representative images of transwell migration assay and I quantitative analysis of migration cells. Scale bar = 100 μm. J Representative images of tube formation assay and K quantitative analysis of total tube length. Scale bar = 100 μm

Article Snippet: Cytoplasmic and nuclear protein fractions were isolated from bEnd.3 cells and freshly isolated cerebral microvessels using the nuclear and cytoplasmic protein extraction Kit (WLA020a, Wanleibio, China).

Techniques: Inhibition, Translocation Assay, Western Blot, Quantitation Assay, Expressing, Control, Derivative Assay, Immunofluorescence, Staining, Diffusion-based Assay, Transwell Migration Assay, Migration, Tube Formation Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Nitric Oxide Donor Alleviates Cardiac Arrest Induced Blood Brain Barrier Injury by Inhibiting HMGB1-ATG5 Mediated Endothelial Autophagy

doi: 10.1007/s10571-026-01706-w

Figure Lengend Snippet: Inhibition of ATG5 improves endothelial function and BBB function without affecting HMGB1 nucleocytoplasmic translocation. A Representative immunoblot images and B quantitation of nuclear HMGB1, cytoplasmic HMGB1, ATG5, LC3B II, LAMP2, occludin and ZO-1 expression in bEnd.3 cells from Control, OGD/R and OGD/R + ATG5 siRNA groups. Histone H3 and β-actin were used as the protein loading control. The images of western blotting data derived from triplicate blots conducted as three independent experiments. C Representative immunofluorescence images of HMGB1 localization in bEnd.3 cells from Control, OGD/R and OGD/R + ATG5 siRNA groups. HMGB1 (green), nuclei stained with DAPI (blue). Scale bar = 50 μm. D Quantification of nuclear and cytoplasmic HMGB1 signals. Nuclear HMGB1 was defined as HMGB1 signal overlapping with DAPI, and cytoplasmic HMGB1 as non-overlapping HMGB1 signal. E Representative images of tube formation assay and F quantitative analysis of total tube length. Scale bar = 100 μm. G Representative images of transwell migration assay and H quantitative analysis of migration cells. Scale bar = 100 μm. I Representative immunoblot images and J quantitation of nuclear HMGB1, cytoplasmic HMGB1, ATG5, LC3B II, LAMP2, occludin and ZO-1 expression in cerebral microvessels of mice from Sham, CA/CPR and CA/CPR + shATG5 groups at 24 h after ROSC. Histone H3 and β-actin were used as the protein loading control. The images of western blotting data derived from triplicate blots conducted as three independent experiments. K Representative immunofluorescence images of occludin and CD31 co-staining in brain sections at 24 h after ROSC and L Representative immunofluorescence images of ZO-1 and CD31 co-staining in brain sections at 24 h after ROSC and M quantification of occludin and CD31 merged area. Scale bars: main panel = 20 μm; zoom-in = 10 μm. N quantification of ZO-1 and CD31 merged area. Scale bars: main panel = 20 μm; zoom-in = 10 μm. O BBB permeability evaluated using EB at 24 h after ROSC. P Brain water content of mice at 24 h after ROSC

Article Snippet: Cytoplasmic and nuclear protein fractions were isolated from bEnd.3 cells and freshly isolated cerebral microvessels using the nuclear and cytoplasmic protein extraction Kit (WLA020a, Wanleibio, China).

Techniques: Inhibition, Translocation Assay, Western Blot, Quantitation Assay, Expressing, Control, Derivative Assay, Immunofluorescence, Staining, Tube Formation Assay, Transwell Migration Assay, Migration, Permeability

Journal: Cellular and Molecular Neurobiology

Article Title: Nitric Oxide Donor Alleviates Cardiac Arrest Induced Blood Brain Barrier Injury by Inhibiting HMGB1-ATG5 Mediated Endothelial Autophagy

doi: 10.1007/s10571-026-01706-w

Figure Lengend Snippet: The protective effect of JS-K against OGD/R-induced endothelial injury depends on the HMGB1-ATG5-mediated autophagy pathway. A Representative immunoblot images and B quantitation of occludin, ZO-1, nuclear HMGB1 and cytoplasmic HMGB1 expression in bEnd.3 cells from Control, OGD/R, OGD/R + JS-K, OGD/R + ATG5 siRNA and OGD/R + JS-K + ATG5 siRNA groups. Histone H3 and β-actin were used as the protein loading control. C The cell viability was examined by CCK-8 assay. D The diffusion rates of FITC-dextran (40 kDa). E Representative fluorescence images of intracellular ROS levels detected by DCFH-DA staining in bEnd.3 cells and F quantification analysis of ROS fluorescence intensity normalized to the Control group. Scale bar = 200 μm. G Representative immunofluorescence images of ZO-1 and occludin in bEnd.3 cells and H quantification of fluorescence intensity for ZO-1 and occludin normalized to the Control group. Scale bar = 50 μm

Article Snippet: Cytoplasmic and nuclear protein fractions were isolated from bEnd.3 cells and freshly isolated cerebral microvessels using the nuclear and cytoplasmic protein extraction Kit (WLA020a, Wanleibio, China).

Techniques: Western Blot, Quantitation Assay, Expressing, Control, CCK-8 Assay, Diffusion-based Assay, Fluorescence, Staining, Immunofluorescence