anti atg5 af647  (R&D Systems)

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: HMGB1 regulates autophagy in endothelial cells through interacting with ATG5. A PPI network functional enrichment analysis of HMGB1 and ATG5. B Molecular docking model showing the predicted interaction interface between HMGB1 (yellow) and ATG5 (blue). The surface structure of the docking complex is displayed on the left. The enlarged panel on the right shows the predicted binding interface and key interacting residues. Hydrogen bonds are indicated by yellow dashed lines. The calculated binding free energy (ΔG) was − 7.9 kcal/mol. C Co-IP analysis of the interaction between HMGB1 and ATG5 in bEnd.3 cells under OGD/R condition. Cell lysates were immunoprecipitated with anti-HMGB1 or anti-ATG5 antibodies, followed by immunoblotting with antibodies against ATG5 or HMGB1. IgG was used as a negative control. D Co-IP of HMGB1 and ATG5 in bEnd.3 cells under different conditions. Cell lysates from Control, OGD/R and OGD/R + Leptomycin B groups were immunoprecipitated with anti-HMGB1 antibody and blotted for ATG5 and HMGB1. IgG was used as a negative control

Article Snippet: For detection of ATG5 in the HMGB1-immunoprecipitated complexes, a mouse anti-ATG5 primary antibody (Mouse, 66744-1-Ig, RRID: AB_2882092, Proteintech) was used, followed by an HRP-conjugated anti-mouse secondary antibody.

Techniques: Functional Assay, Binding Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Negative Control, Control

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: For detection of ATG5 in the HMGB1-immunoprecipitated complexes, a mouse anti-ATG5 primary antibody (Mouse, 66744-1-Ig, RRID: AB_2882092, Proteintech) was used, followed by an HRP-conjugated anti-mouse secondary antibody.

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: For detection of ATG5 in the HMGB1-immunoprecipitated complexes, a mouse anti-ATG5 primary antibody (Mouse, 66744-1-Ig, RRID: AB_2882092, Proteintech) was used, followed by an HRP-conjugated anti-mouse secondary antibody.

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

Journal: iScience

Article Title: Lithium attenuates HIV-1 latency reversal in an autophagy-independent way

doi: 10.1016/j.isci.2025.114085

Figure Lengend Snippet: Lithium induces autophagy in CD4 + T J-lat 10.6 cells J-lat 10.6, CD4 + T cells, were treated with torin1 (TO) and lithium (10–30 mM) for 24 h and collected for (A) flow cytometry and (B) western blotting. (A) Samples were stained with rabbit anti-LC3B, followed by anti-rabbit AF594. Graph shows data as a relative percentage of untreated (UT) sample. On the right, is shown a representative histogram comparing the shift in MFI between treatments. (B) Lysates were immunoblotted for p62 as an indirect measure of autophagic flux, and β-actin was used as a loading control. (C and D) Samples were treated as in (A-B), and 2 h before collection, the samples were transferred to wells containing coverslips treated with poly-D-lysine and incubated with 50 nM Bafilomycin A1 to evaluate autophagy. Coverslips were then fixed and processed for immunofluorescence and stained against LC3B and Lamp-1, followed by the 2 nd Antibodies AF647 and AF594, respectively. (C) Graph shows the number of autophagosomes per cell in each condition quantified through co-localization of LC3B-II and Lamp-1 (see methodology for details). (D) Representative confocal images showing individual channels (AF647 and AF594) and the merge with DAPI. The large inset is the representative data showed in (C), was obtained by zooming in the selected area and shows only the co-localization (∩) of LC3BII and Lamp-1 as a readout for autophagosomes. (D) Large inset, scale bars represent 10 μm; small inset, scale bars represent 20 μm. (A–C) Bars represent the mean ± SEM. (A) n = 3, (B) n = 5. (D) Data from one experiment ( n = 2). (A and B) ANOVA one-way, Dunnett’s post-test. (C) ANOVA two-way, Tukey’s post-test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Antibodies concentration: 1:50 mouse anti-LC3B AF594 (Novus Biologicals); 1:100 rabbit anti-LC3B (Abcam) 1:100 anti-ATG5 AF647 (R&D systems), 1:100 anti-Beclin1 AF405 (Novus Biologicals), 1:100 anti-NF-kB/p65 p-S529 (Biolegend), 1:200 Donkey anti-mouse AF594 (Invitrogen).

Techniques: Flow Cytometry, Western Blot, Staining, Control, Incubation, Immunofluorescence

Journal: iScience

Article Title: Lithium attenuates HIV-1 latency reversal in an autophagy-independent way

doi: 10.1016/j.isci.2025.114085

Figure Lengend Snippet: Induction of autophagy favors latency reversal (A) Diagram of the experimental setup. shRNAs against ATG5, Beclin-1 and the non-target control (NT) were packed into lentivirus particles and transduced into J-lat 10.6 cells. three target sequences for ATG5 and three for BECN1 were selected, labeled as shATG5 #1, #2, and #3, and shBECN1 #1, #2, and #3, respectively. After selection with puromycin, the cells were expanded, and a reactivation assay was performed. Only (B, D, and F) shATG5 #1 and (C, E and G) shBECN1 #2 are shown. The remaining targets are shown in supplementary figures ( and ). (B and C) Knockdown was initially confirmed by western blotting. (D–G) The transduced cells were treated with lithium or torin1 for 24 h and then reactivated with (D and E) TNF-α or (F and G) PMA for another 24 h. Samples were processed for flow cytometry and stained for (C and D) ATG5-AF647 or (F andG) beclin1-AF405. Samples were acquired using the spectral flow cytometer (Sony ID7000). HIV-1 reactivation was analyzed in cells knocked down for ATG5 or Beclin-1 by gating in cells with lower expression of ATG5 or beclin-1 and then compared to the non-targeting control (not gated in ATG5 or beclin-1). Data were normalized to the untreated samples from non-target (NT)-transduced cells. The group “not reactivated” represents the negative control. See also and . Bars represent mean ± SEM. n = 3, in triplicates. two-way ANOVA and Tukey’s multiple comparison test. ∗ p < 0.05; ∗∗ p < 0.001; ns, non-significant.

Article Snippet: Antibodies concentration: 1:50 mouse anti-LC3B AF594 (Novus Biologicals); 1:100 rabbit anti-LC3B (Abcam) 1:100 anti-ATG5 AF647 (R&D systems), 1:100 anti-Beclin1 AF405 (Novus Biologicals), 1:100 anti-NF-kB/p65 p-S529 (Biolegend), 1:200 Donkey anti-mouse AF594 (Invitrogen).

Techniques: Control, Labeling, Selection, Knockdown, Western Blot, Flow Cytometry, Staining, Expressing, Negative Control, Comparison

Journal: Scientific Reports

Article Title: Immune cell diversity and regenerative markers reveal interactions among macrophages, rodlet cells, and stem cells in the kidney of Poecilia sphenops

doi: 10.1038/s41598-025-11679-3

Figure Lengend Snippet: Immunohistochemistry of APG5. ( A , B ) Macrophages (arrowheads) express APG5. ( C ) Rodlet cells (arrowheads) express APG5 around renal tubules (RT). ( D ) Podocytes (arrowheads) express APG5 in the renal corpuscle (RC).

Article Snippet: The sections were exposed to diluted (1:100) primary antibodies against the rabbit polyclonal S100 protein for an entire night at 4 °C (Z0311, Dako, Glostrup, Denmark), mouse monoclonal anti-CD68 (Santa Cruz, sc-17832), rabbit polyclonal Nicotinic Acetylcholine R alpha 7 NACHRA7 (ABclonal, A7844), rabbit polyclonal interleukin 1 beta (IL-1β) (sc-7884, Santa Cruz Biotechnology, Heidelberg Germany), rabbit polyclonal iNOS-2 (RB-1605, Thermo Fisher Scientific, UK), mouse monoclonal autophagy protein 5 (APG5) (sc-133158, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit polyclonal nuclear factor kappa B (NF-κB) (10745-1-AP, Proteintech, USA), rabbit polyclonal nuclear factor erythroid 2-related factor 2 (Nrf2) (sc-722, Santa Cruz Biotechnology, Heidelberg, Germany), and rabbit polyclonal SRY-Box transcription factor 9 (Sox9) (AB5535, Sigma-Aldrich, Madrid, Spain).

Techniques: Immunohistochemistry