Anti-Transferrin antibody - Serum Loading Control

• WB

AbReview32187****

Western blot - Anti-Transferrin antibody - Serum Loading Control (AB82411)

All lanes:

Western blot - Anti-Transferrin antibody - Serum Loading Control (ab82411) at 1/1000 dilution

All lanes:

Mouse pancreatic acinar cells at 30 µg

Secondary

All lanes:

Goat anti-rabbit IgG (HRP conjugated) at 1/5000 dilution

Predicted band size: 77 kDa

true

Exposure time: 10min

This image is courtesy of an anonymous Abreview.

• WB

Unknown

Western blot - Anti-Transferrin antibody - Serum Loading Control (AB82411)

All lanes:

Western blot - Anti-Transferrin antibody - Serum Loading Control (ab82411) at 1/10000 dilution

All lanes:

rat apotransferrin

Predicted band size: 77 kDa

Observed band size: 77 kDa

true

• WB

CiteAb

Western blot - Anti-Transferrin antibody - Serum Loading Control (AB82411)

Western Blotting using Anti-Transferrin antibody, ab82411. Publication image from Perdomo, J. et al., 2019, Nat Commun, 30899022. Legend direct from paper.

NETs are present in HIT patients. a cfDNA in HIT patients’ plasma (n = 21) relative to normal controls (n = 18) was detected using PicoGreen dsDNA fluorescence assay. b MPO levels in HIT patients’ plasma (n = 21) and normal controls (n = 18) were measured by ELISA. c Neutrophil elastase concentration in patients’ plasma (n = 20) relative to normal controls (n = 18) and d CitH3 levels in HIT patients’ plasma (n = 21) relative to normal controls (n = 18) was determined by ELISA. e Western blot images of CitH3 probed with anti-CitH3 antibody in normal controls and HIT patients’ plasma. Each lane represents a different donor’s plasma. Transferrin (Transf) was used as a loading control. Arrowhead indicates CitH3 band. Arrow denotes transferrin. f Representative flow cytometry density plots using fresh blood backgated for neutrophils (CD15+ CD16+ population shown in g). g Flow cytometric determination of neutrophils (CD15+ CD16+ population, Neut.). LDGs within the Neut population are shown. h Neutrophil–platelet aggregates (CD41+ events within the CD15+ CD16+ population). The graph shows the quantification of the flow cytometry data shown on the left in healthy controls (n = 10) and HIT patients (n = 3). NPA, neutrophil–platelet aggregates. i Representative dotplot of NETs present in vivo in healthy controls (left panels) and HIT patients (middle panel). The numbers in the quadrants indicate percentage of gated events. NETs were defined as CitH3 and MPO double positive events within the CD15+ CD16+ population. The graph shows the quantification of the flow cytometry data shown on the left in healthy controls (n = 10) and HIT patients (n = 3). Statistics, Mann–Whitney test. *P < 0.05; **P < 0.01; ****P < 0.0001. Mean ± s.e.m. shown in all graphs. LDG, low-density granulocytes, Neut, neutrophils. Source data for (a, b, c, d, e, h, i) are provided as a Source Data file

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• WB

CiteAb

Western blot - Anti-Transferrin antibody - Serum Loading Control (AB82411)

Western Blotting using Anti-Transferrin antibody, ab82411. Publication image from Hackl, M. T. et al., 2019, Nat Commun, 31222048. Legend direct from paper.

Brain leptin signaling increases liver TG secretion and reduces hepatic steatosis. a Protocol for acute ICV leptin infusion experiments. b Plasma TG accumulation in ICV leptin/vehicle-infused rats after a tyloxapol bolus injection (Leptin : 1 µg/h; n ≥ 12 per group). c VLDL secretion rate calculated from the slopes depicted in Fig. 1b. d Western blot of ApoB100 and ApoB48 in plasma samples at timepoint 180 min from acute ICV leptin/vehicle infusion experiments. e Quantification of the Western blot analysis from Fig. 1d (n ≥ 7 per group). f Protocol for chronic ICV leptin/vehicle experiments. g Body weights and h hepatic lipid content assessed by 1H-MRS after 28 days of chronic ICV leptin/vehicle infusion (Leptin : 0.3 µg/day). i Western blot analysis of ApoB100 and ApoB48 from plasma after chronic leptin/vehicle infusion collected at the end of the experiment. j Quantification of the Western blot analysis in Fig. 1i (n = 8 per group). k Relative changes compared to baseline in hepatic lipid content after 28 days of chronic ICV leptin/vehicle infusion. l Protocol for chronic ICV leptin receptor antagonist experiments. m Relative changes in hepatic lipid content assessed by 1H-MRS during 28 days of blocking endogenous leptin signaling with an ICV infused peptide leptin receptor antagonist (Leptin receptor antagonist : 6 µg/day; n = 5 per group). All data are mean ± SEM; *p < 0.05; **p < 0.01; vs vehicle group by two-tailed Student’s t test; open circles : ICV vehicle; black squares : ICV leptin except for (m) : ICV leptin receptor antagonist

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• WB

CiteAb

Western blot - Anti-Transferrin antibody - Serum Loading Control (AB82411)

Western Blotting using Anti-Transferrin antibody, ab82411. Publication image from Hackl, M. T. et al., 2019, Nat Commun, 31222048. Legend direct from paper.

Brain leptin signaling increases liver TG secretion and reduces hepatic steatosis. a Protocol for acute ICV leptin infusion experiments. b Plasma TG accumulation in ICV leptin/vehicle-infused rats after a tyloxapol bolus injection (Leptin : 1 µg/h; n ≥ 12 per group). c VLDL secretion rate calculated from the slopes depicted in Fig. 1b. d Western blot of ApoB100 and ApoB48 in plasma samples at timepoint 180 min from acute ICV leptin/vehicle infusion experiments. e Quantification of the Western blot analysis from Fig. 1d (n ≥ 7 per group). f Protocol for chronic ICV leptin/vehicle experiments. g Body weights and h hepatic lipid content assessed by 1H-MRS after 28 days of chronic ICV leptin/vehicle infusion (Leptin : 0.3 µg/day). i Western blot analysis of ApoB100 and ApoB48 from plasma after chronic leptin/vehicle infusion collected at the end of the experiment. j Quantification of the Western blot analysis in Fig. 1i (n = 8 per group). k Relative changes compared to baseline in hepatic lipid content after 28 days of chronic ICV leptin/vehicle infusion. l Protocol for chronic ICV leptin receptor antagonist experiments. m Relative changes in hepatic lipid content assessed by 1H-MRS during 28 days of blocking endogenous leptin signaling with an ICV infused peptide leptin receptor antagonist (Leptin receptor antagonist : 6 µg/day; n = 5 per group). All data are mean ± SEM; *p < 0.05; **p < 0.01; vs vehicle group by two-tailed Student’s t test; open circles : ICV vehicle; black squares : ICV leptin except for (m) : ICV leptin receptor antagonist

false

• WB

CiteAb

Western blot - Anti-Transferrin antibody - Serum Loading Control (AB82411)

Western Blotting using Anti-Transferrin antibody, ab82411. Publication image from Perdomo, J. et al., 2019, Nat Commun, 30899022. Legend direct from paper.

NETosis is required for thrombus formation in HIT. a Neutrophils plus platelets were treated with PF4, heparin and normal IgG (n = 4, orange), or HIT IgG plus heparin without (n = 3, red) or with GSK484 (n = 5, green) or IV.3 antibody (n = 4, magenta). Treated neutrophils alone are also shown (n = 4, blue). The extracellular DNA release determined as in Fig. 4b. Mean ± s.e.m. b Quantification of neutrophil–platelet aggregates, mean ± s.e.m. c NETs after 5 h treatment with antibodies and inhibitors, mean ± s.e.m. Fluorescent microscopy images of thrombi in microchannels. d Blood pre-treated with KKO plus heparin plus vehicle control, DNase I or IV.3. Neutrophils (anti-CD15 AF594, left panel, blue), nucleated cells (Hoechst, middle panel, blue), the extracellular DNA (Sytox green, green) and platelets (anti-CD41 AF647, magenta). Scale bar : 50 µm. e Area coverage of neutrophils, DNA and platelets. n = 3, mean ± s.d. f Whole blood (WB) (left panels), neutrophil (Neut) depleted blood (middle panels) or depleted blood reconstituted (recons) with autologous neutrophils (right panels) incubated with KKO and heparin, treated as described in (d). Plt, platelets; Scale bar : 50 µm. g Quantification as in (e), n = 3, mean ± s.d. hFcγRIIa+/hPF4+ mice treated with normal (n = 5, black) or HIT IgG plus heparin plus vehicle control (n = 7, dotted red) or anti-CD62p (n = 3, green), agIV.3 antibody (n = 8, blue), GSK484 (n = 8, magenta) or DNase I (n = 8, grey). Mean ± s.e.m. i Box-and-whiskers plot of cfDNA in mouse plasma described in (h) (n = 4 for normal IgG, anti-CD62p; n = 5 for DNase I; n = 6 for vehicle, IV.3, GSK484). Middle line, bounds of box and whiskers represent the median, 25th to 75th percentiles, and minimum and maximum values, respectively. j Mouse lungs imaged as described in (3b) and plot of fluorescence intensity. Mean ± s.e.m. k Representative western blots of mouse plasma described in (h) probed with anti-CitH3 antibody. Arrowhead, CitH3. Arrow, transferrin (transf, loading control). Statistics : (b, c, h, i, j) Kruskal–Wallis test for comparison of groups with versus without inhibitor. P-values adjusted relative to HIT IgG (vehicle). e, g one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Source data for (a, b, c, e, g, h, i, j, k) are provided as a Source Data file

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• WB

CiteAb

Western blot - Anti-Transferrin antibody - Serum Loading Control (AB82411)

Western Blotting using Anti-Transferrin antibody, ab82411. Publication image from Perdomo, J. et al., 2019, Nat Commun, 30899022. Legend direct from paper.

HIT IgG induces thrombosis and NETs in a HIT mouse model. aFcγRIIa+/hPF4+ mice were injected with KKO or control mouse IgG (n = 3, left panel) or HIT patients’ IgG or normal IgG (n = 4, right panel). Heparin administered at 1U/g. Platelet percentage was calculated relative to basal levels. b Images of mouse lungs harvested 5 h after treatment. Green fluorescence indicates anti-CD42c Dylight 649 platelet clots in lungs. Graph of lung fluorescence from mice treated in (a) (n = 4. For IgG1-3, n = 5). c Lung sections from mice treated as in (b). Upper panels show platelet-rich thrombi (magenta) which are present in all cases except with normal IgG. Blue, nuclei. Lower panels, H&E staining. Arrows indicate clots. Arrowheads show small clots in KKO-treated mice. 10X objective. Scale bar, 100 µm. d Carstair’s staining of KKO- (upper panel) or HIT IgG- (lower panel) induced thrombi in mouse lungs. Fibrin (orange–red, arrows); leucocytes (dark blue, yellow arrowheads); red blood cells (yellow–red, green arrowheads). Platelets (grey–blue) are mixed with fibrin. Scale bar : 20 µm. e cfDNA and MPO activity. Fold change in cfDNA in mouse plasma 1 h or 3 h after treatment with KKO (n = 10) or control mouse IgG (mIgG, n = 4) (left panel) or patient’s HIT IgG (n = 5) or normal control (n = 3, middle panel). Ratio of MPO activity at 3 h relative to time 0 following treatment with HIT IgG (n = 9) or normal control (n = 4, right panel). f Representative western blots of CitH3 in plasma from mice treated in (a). Arrowhead; CitH3. Arrow; transferrin (transf, loading control). Dotted lines : removal of irrelevant lanes. g Thrombi in mouse lung imaged by confocal microscopy. Platelets (magenta), nucleated cells (blue), MPO (green). Scale bar : 20 µm. h Magnified details of dotted area in g of nuclei (arrowhead) and decondensed neutrophil nucleus (arrow). Other panels denote MPO, platelets and overlay images, respectively. Scale bar : 10 µm. i Neutrophils were stained with Ly6G (green), CitH3 (magenta) and DNA (blue). Arrow, extracellular DNA; arrowhead, CitH3. Scale bar : 20 µm. Statistical analyses, (a, e) Mann–Whitney test. b Kruskal–Wallis test. p-values adjusted relative to normal IgG. Mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Source data for (a, b, e, f) are provided as a Source Data file

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