rabbit anti flap  (Novus Biologicals)

Journal: bioRxiv

Article Title: Neutrophils secrete exosome-associated DNA to resolve sterile acute inflammation

doi: 10.1101/2024.04.21.590456

Figure Lengend Snippet: a. An overlay image of PMNs on fibrinogen-fibronectin coated Quantifoil grids stimulated with 100 nM LTB 4 for 15 min in the presence of SYTOXgreen, plunge-frozen, and imaged on Leica Stellaris 5 cryo-confocal, showing the reflected light (gray) and extracellular DNA (green). Dashed yellow lines highlight PMNs on the grid square. The scale bar is 50 μm. b. A low-magnification TEM image (6500x) of the highlighted region (red box) in panel a, is overlaid with the SYTOX green fluorescence signal. The cyan box indicates the area where cryo-ET data was acquired. The scale bar is 500 nm. c. A slice through the tomogram of the region highlighted (cyan box) in panel b, with insets showing ‘bead-on-string’ like structures (yellow arrowheads). The scale bar is 100 nm. d. Segmentation of vesicles (<100 nm in orange, >100 nm in olive green) and the DNA (cyan) corresponding to the tomogram in panel c. The scale bar is 100 nm. See associated Movie 1. e. Representative western blot of exosomes obtained from fractions 4-9 of 5-40% density gradient centrifugation from PMNs stimulated with 100 nM LTB 4 and treated with 50 µg/ml DNase I followed by immunoblotting for FLAP, 5LO, and histone H3. N=3. f. Bar graph showing the quantifications of the band intensity of FLAP, 5LO, and histone H3, in control and DNase I treated exosome samples. Data points (red dots) from 3 independent experiments were plotted as mean ± s.e.m. P values determined using two-way ANOVA are reported. g. Schematic illustration of DNA packaging and secretion during the nuclear budding and the fusion of NE-MVB with the PM. Note the change in the orientation of the LBR N-terminal Tudor domain as it moves from the inner nuclear membrane to the PM.

Article Snippet: The blocked tissue sections were stained using primary antibodies: chicken anti-keratin 14 (Biolegend #906004, 1:1000), rabbit anti-citrullinated histone H3 (Abcam #5103, 1:800), rabbit anti-FLAP (Invitrogen #MA5-37933, 1:200) and mouse anti-dsDNA (Santacruz Biotechnology #58749, 1:100) in blocking solution overnight at 4 °C.

Techniques: Fluorescence, Tomography, Western Blot, Gradient Centrifugation, Membrane

Journal: bioRxiv

Article Title: Neutrophils secrete exosome-associated DNA to resolve sterile acute inflammation

doi: 10.1101/2024.04.21.590456

Figure Lengend Snippet: a. Representative western blot images showing the levels of CD63, FLAP, LBR, and histone H3 expression in different subcellular fractions obtained by density-gradient ultracentrifugation of cytoplasmic contents of LMNA and LMNA/LBR KO dHL60 cells treated with LTB 4 (100 nM) for 30 min. The red box highlights the MVB fractions. Molecular weights - in kiloDaltons (kDa) - are located on the left side of the panels. N=3. b. Bar graphs showing the intensity levels of proteins quantified using western blot images as shown in panel a. Data is plotted as mean ± s.e.m. N=3. c. Representative western blot images showing the levels of LBR, histone H3, 5LO, and FLAP intensities in the cell lysates and purified exosomes obtained from LMNA KO and LMNA/LBR KO dHL60 cells treated with LTB 4 (100 nM) for 30 min. Molecular weights in kDa are located on the left side of the panels. N=3. d. Bar graphs showing changes in the intensity levels of proteins in exosome fractions as shown in panel c. Data is plotted as mean ± s.e.m. and P values calculated using two-way ANOVA analysis are shown. N=3. e. Histogram showing the intensity of surface LBR staining in SCR dHL60 cells treated with either DMSO or 100 nM LTB 4 and immunostained for LBR, under non-permeabilizing conditions. Fixed, TritonX-100 permeabilized SCR cells were used as a positive control to stain intracellular LBR, and LBR KO cells were used as a negative control. f. Scatter dot plot showing the percentage of LBR (surface)-positive SCR dHL60 cells post LTB 4 activation. Datapoints from the same experiments are similarly color-coded and are plotted as mean ± s.e.m. and P values calculated using paired t-tests are shown. N=3. g. Scatter dot plot showing the relative change in the surface LBR intensity within indicates conditions (x-axis). Datapoints from the same experiments are similarly color-coded and are plotted as mean ± s.e.m. and P values calculated using RM one-way ANOVA are shown. N=3. h. Scatter dot plot showing the percent of DNA-secreting cells within SCR, LMNA KO, and LMNA/LBR KO dHL60 cells migrating towards LTB 4 , within an observation window of 40,000 μm 2 for 1 hr. Datapoints from the same experiments are similarly color-coded and are plotted as mean ± s.e.m., and P values obtained using paired t-tests are shown. N=5. See associated Movie 8.

Article Snippet: The blocked tissue sections were stained using primary antibodies: chicken anti-keratin 14 (Biolegend #906004, 1:1000), rabbit anti-citrullinated histone H3 (Abcam #5103, 1:800), rabbit anti-FLAP (Invitrogen #MA5-37933, 1:200) and mouse anti-dsDNA (Santacruz Biotechnology #58749, 1:100) in blocking solution overnight at 4 °C.

Techniques: Western Blot, Expressing, Purification, Staining, Positive Control, Negative Control, Activation Assay

Journal: bioRxiv

Article Title: Neutrophils secrete exosome-associated DNA to resolve sterile acute inflammation

doi: 10.1101/2024.04.21.590456

Figure Lengend Snippet: a. Representative Airyscan microscopy images of TPA-treated (12h) ear cryosections (20 µm) from mice injected with either 100 μg DNase I or PBS, showing FLAP-dsDNA PLA signal (magenta dots) and DAPI (gray, nuclei). Images are presented as a ‘sum of slices’ projection of z-stacks. The scale is 100 μm and it is 20 µm in the insets. N=3. b. Scatter dot plot showing total FLAP-dsDNA PLA dots in the ear post 12 h TPA treatment. The maximum intensity projection images of 20 µm cryosections were calculated by object segmentation using CellProfiler and data points (red circles) pooled from 3 independent experiments are presented as mean ± s.e.m. P values calculated using the Mann-Whitney test are shown. c. Graph showing the four-parameter logistic curve fit (thick lines) of normalized PLA, Ly6G, and citH3 intensity distribution from the ventral to the dorsal side in inflamed ear post 12 h TPA treatment. The wavy lines represent the mean of 7 datapoints pooled from 3 independent experiments. d. Bar graph showing the fold change in the expression of various PPARα-responsive genes in dHL60 cells. Datapoints (black open circles) are plotted as mean ± s.e.m. and P values quantified using two-way ANOVA are shown. N=3. e-f. Representative Airyscan microscopy images of 20 µm thick cryosections of TPA-treated ear under different conditions as mentioned on the right side of the respective panels showing the relative localization of Ly6G (magenta, neutrophils), K14 (cyan, epidermis), and DAPI (gray, nuclei). The tiled z-stacks are presented here as a ‘sum of slices’ projection. The scale is 100 μm. N=3. g-h. Scatter dot plots showing the changes in ear thickness and Ly6G intensity per unit area within the dermis of inflamed ears under different conditions at 12 and 24 hrs post-TPA treatment, as shown in panels e-f. Datapoints (circle/dots) pooled from 3 independent experiments are plotted as mean ± s.e.m. and P values calculated using two-way ANOVA analysis are shown on the graph.

Article Snippet: The blocked tissue sections were stained using primary antibodies: chicken anti-keratin 14 (Biolegend #906004, 1:1000), rabbit anti-citrullinated histone H3 (Abcam #5103, 1:800), rabbit anti-FLAP (Invitrogen #MA5-37933, 1:200) and mouse anti-dsDNA (Santacruz Biotechnology #58749, 1:100) in blocking solution overnight at 4 °C.

Techniques: Microscopy, Injection, MANN-WHITNEY, Expressing