Anti-ABCA1 antibody [AB.H10]

• Flow Cyt

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Flow Cytometry - Anti-ABCA1 antibody [AB.H10] (AB18180)

Overlay histogram showing HepG2 cells stained with ab18180 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab18180, 2μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.

• WB

Supplier Data

Western blot - Anti-ABCA1 antibody [AB.H10] (AB18180)

The lower bands are from the secondary anti-mouse antibody reacting to the endogenous mouse antibodies from the tissue.

All lanes:

Western blot - Anti-ABCA1 antibody [AB.H10] (ab18180) at 1/1000 dilution

Lanes 1, 3 and 5:

Wild type mouse liver tissue lysate at 100 µg

Lanes 2, 4 and 6:

Knockout mouse liver tissue lysate at 100 µg

Predicted band size: 254 kDa

Observed band size: 254 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ABCA1 antibody [AB.H10] (AB18180)

Immunoreactivity of pancreas tissue for ABCA1 protein. Left panel : strong immunopositivity of exocrine glandular cells of the pancreas, especially in the basal region (arrows). Interstitial microvascular cells are apparently negative. Cells within the islet were weakly positive (data not shown). Right panel : the negative control. Hematoxylin counterstain.

• WB

CiteAb

Western blot - Anti-ABCA1 antibody [AB.H10] (AB18180)

Western Blotting using Anti-ABCA1 antibody [AB.H10]Anti-ABCA1 antibody [AB.H10], ab18180. Publication image from Rao, G. N. et al., 2015, Nat Commun, 26104863. Legend direct from paper.

The lack of Pak1 diminishes monocyte adhesion to the aorta.(a) Aortas from 24-week-old ApoE−/− mice were incubated with BCECF-labelled monocytes from C57BL/6 and Pak1−/− mice, and the adherent monocytes were observed under a fluorescent microscope and counted. (b) Aortas from ApoE−/− and ApoE−/− : Pak1−/− mice fed with WD for 16 weeks were incubated with BCECF-labelled monocytes from C57BL/6 mice and the adherent cells were observed under a fluorescent microscope and counted. (c) Peritoneal macrophages from WT, ApoE−/− and Pak1−/− mice were treated with and without cholesterol (40 µg ml−1), IL-6 (20 ng ml−1) or MCP-1 (50 ng ml−1) for 6 h and cell extracts were prepared. Equal amounts of protein from control and each treatment were analysed by western blotting for CD36 and ABCA1 levels using their specific antibodies and normalized for β-tubulin. Bar graphs in a and b represent three independent experiments with three mice per group per experiment. Data were presented as mean±s.d. and assessed by Student's t-test. *P<0.01 versus WT or ApoE−/− mice. Scale bars, 20 µm (a,b).

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• WB

CiteAb

Western blot - Anti-ABCA1 antibody [AB.H10] (AB18180)

Western Blotting using Anti-ABCA1 antibody [AB.H10]Anti-ABCA1 antibody [AB.H10], ab18180. Publication image from Rao, G. N. et al., 2015, Nat Commun, 26104863. Legend direct from paper.

Pak1 regulates SMC migration and lesion formation.Aortic SMCs from ApoE−/− and ApoE−/− : Pak1−/− mice fed with WD for 16 weeks were isolated, cultured and tested for their purity using anti-SMCα-actin antibodies (a), analysed for IL-6 expression by FACS (b) and subjected to wound-healing migration assay (c). (d) Aortic SMC extracts from ApoE−/− and ApoE−/− : Pak1−/− mice were analysed by western blotting for the indicated proteins using their specific antibodies. FACS analysis of CD36 expression (e) and Dil-OxLDL uptake (f) are shown. SMCs isolated from ApoE−/− and ApoE−/− : Pak1−/− mice fed with WD for 16 weeks were either incubated with OxLDL or Dil-OxLDL for 6 h and stained with Oil red O (g, left panel) or observed under fluorescent microscope (g, right panel), or subjected to cholesterol efflux (h) as described in Methods. (i) Extracts of SMCs isolated from WT, Pak1−/− and ApoE−/− mice fed with CD were analysed by western blotting for the indicated proteins using their specific antibodies. (j) SMCs from WT and Pak1−/− mice fed with CD were treated with OxLDL (10 µg ml−1) for the indicated time periods and RNA was isolated and analysed by qRT–PCR for IL-6 and MCP-1 levels. Bar graph in c represents quantification of three independent experiments. Bar graph in d represents the quantification of three western blottings each from a group of two pooled arteries. Data were presented as mean±s.d. and assessed by Student's t-test. *P<0.01 versus ApoE−/− mice; †P<0.01 versus WT vehicle control; #P<0.01 versus WT mice+OxLDL. Scale bars, 50 µm (a) and 100 and 50 µm (g, left and right panels, respectively).

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• WB

CiteAb

Western blot - Anti-ABCA1 antibody [AB.H10] (AB18180)

Western Blotting using Anti-ABCA1 antibody [AB.H10]Anti-ABCA1 antibody [AB.H10], ab18180. Publication image from Rao, G. N. et al., 2015, Nat Commun, 26104863. Legend direct from paper.

Pak1 regulates SMC migration and lesion formation.Aortic SMCs from ApoE−/− and ApoE−/− : Pak1−/− mice fed with WD for 16 weeks were isolated, cultured and tested for their purity using anti-SMCα-actin antibodies (a), analysed for IL-6 expression by FACS (b) and subjected to wound-healing migration assay (c). (d) Aortic SMC extracts from ApoE−/− and ApoE−/− : Pak1−/− mice were analysed by western blotting for the indicated proteins using their specific antibodies. FACS analysis of CD36 expression (e) and Dil-OxLDL uptake (f) are shown. SMCs isolated from ApoE−/− and ApoE−/− : Pak1−/− mice fed with WD for 16 weeks were either incubated with OxLDL or Dil-OxLDL for 6 h and stained with Oil red O (g, left panel) or observed under fluorescent microscope (g, right panel), or subjected to cholesterol efflux (h) as described in Methods. (i) Extracts of SMCs isolated from WT, Pak1−/− and ApoE−/− mice fed with CD were analysed by western blotting for the indicated proteins using their specific antibodies. (j) SMCs from WT and Pak1−/− mice fed with CD were treated with OxLDL (10 µg ml−1) for the indicated time periods and RNA was isolated and analysed by qRT–PCR for IL-6 and MCP-1 levels. Bar graph in c represents quantification of three independent experiments. Bar graph in d represents the quantification of three western blottings each from a group of two pooled arteries. Data were presented as mean±s.d. and assessed by Student's t-test. *P<0.01 versus ApoE−/− mice; †P<0.01 versus WT vehicle control; #P<0.01 versus WT mice+OxLDL. Scale bars, 50 µm (a) and 100 and 50 µm (g, left and right panels, respectively).

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• WB

CiteAb

Western blot - Anti-ABCA1 antibody [AB.H10] (AB18180)

Western Blotting using Anti-ABCA1 antibody [AB.H10]Anti-ABCA1 antibody [AB.H10], ab18180. Publication image from Fernández-Hernando, C. et al., 2016, Nat Commun, 27460411. Legend direct from paper.

ANGPTL4 deficiency promotes macrophage foam cell formation and apoptosis.(a) Representative pictures from WT and Angptl4−/− mouse peritoneal macrophages incubated with or without Ac- LDL (120 µg ml−1) for 24 h and stained with BODIPY 493/503 (1 µg ml−1) and DAPI (Green and blue, respectively). Scale bar, 5 µm. Quantification of the mean average intensity is in the right panel. (b) Total cholesterol content in peritoneal macrophages isolated from WT and Angptl4−/− mice incubated with or without Ac-LDL (120 µg ml−1) for 24 h. (c) Flow cytometry analysis of DiI-Ox-LDL binding in peritoneal macrophages incubated with DiI-Ox-LDL (30 µg cholesterol per ml) for 30 min at 4 °C. At the end of the incubation period, cells were washed and incubated in RPMI 10% FBS media for 15 min at 37 °C to allow the internalization. (d) Flow cytometry analysis of DiI-Ox-LDL uptake in peritoneal macrophages incubated with DiI-Ox-LDL (30 µg cholesterol per ml) for 2 h at 37 °C. The results are expressed in terms of specific MFI after subtracting auto-fluorescence of cells incubated in the absence of DiI-Ox-LDL. (e) Cholesterol efflux to apolipoprotein A1 (ApoA1) in peritoneal macrophages isolated from WT and Angptl4−/− mice stimulated with or without T0901317 (T090). (f) Western blot analysis of indicated proteins in peritoneal macrophages from WT and Angptl4−/− mice incubated with or without Ac-LDL (120 µg ml−1) for 24 h. (g) Western blot analysis (representative of three blots) of ABCA1 expression in WT and Angptl4−/− peritoneal macrophages incubated with Ac-LDL for 24 h. Surface ABCA1 was isolated using biotinylation followed by incubation with neutravidin. HSP90 is used as loading control (f and g). Full scans of westerns blots are provided in Supplementary Fig. 8. (h) Representative confocal images of mouse peritoneal macrophages from WT and Angptl4−/− mice incubated with Ac-LDL for 24 h and stained with cholera toxin B (CTxB), ABCA1 and DAPI. Quantification of co-localization of CTxB and ABCA1 is on the right panel. Scale bar, 10 µm. (i) Representative images of WT and Angptl4−/− macrophages cultured on coverslips and treated with or without Ac-LDL (120 µg ml−1) in combination with ACAT inhibitor (58035) for 24 h to induce lipid-loading-induced apoptosis (scale bars, 200 µm). Apoptosis was detected using Annexin-V staining. Right panel shows the quantification of percentage of apoptotic cells from four random fields from each cover slip. All data represent the mean±s.e.m. from at least three experiments in duplicate; *P<0.05 compared with WT macrophages by unpaired t-test. MFI, median intensity of fluorescence.

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• WB

CiteAb

Western blot - Anti-ABCA1 antibody [AB.H10] (AB18180)

Western Blotting using Anti-ABCA1 antibody [AB.H10]Anti-ABCA1 antibody [AB.H10], ab18180. Publication image from Fernández-Hernando, C. et al., 2016, Nat Commun, 27460411. Legend direct from paper.

ANGPTL4 deficiency promotes macrophage foam cell formation and apoptosis.(a) Representative pictures from WT and Angptl4−/− mouse peritoneal macrophages incubated with or without Ac- LDL (120 µg ml−1) for 24 h and stained with BODIPY 493/503 (1 µg ml−1) and DAPI (Green and blue, respectively). Scale bar, 5 µm. Quantification of the mean average intensity is in the right panel. (b) Total cholesterol content in peritoneal macrophages isolated from WT and Angptl4−/− mice incubated with or without Ac-LDL (120 µg ml−1) for 24 h. (c) Flow cytometry analysis of DiI-Ox-LDL binding in peritoneal macrophages incubated with DiI-Ox-LDL (30 µg cholesterol per ml) for 30 min at 4 °C. At the end of the incubation period, cells were washed and incubated in RPMI 10% FBS media for 15 min at 37 °C to allow the internalization. (d) Flow cytometry analysis of DiI-Ox-LDL uptake in peritoneal macrophages incubated with DiI-Ox-LDL (30 µg cholesterol per ml) for 2 h at 37 °C. The results are expressed in terms of specific MFI after subtracting auto-fluorescence of cells incubated in the absence of DiI-Ox-LDL. (e) Cholesterol efflux to apolipoprotein A1 (ApoA1) in peritoneal macrophages isolated from WT and Angptl4−/− mice stimulated with or without T0901317 (T090). (f) Western blot analysis of indicated proteins in peritoneal macrophages from WT and Angptl4−/− mice incubated with or without Ac-LDL (120 µg ml−1) for 24 h. (g) Western blot analysis (representative of three blots) of ABCA1 expression in WT and Angptl4−/− peritoneal macrophages incubated with Ac-LDL for 24 h. Surface ABCA1 was isolated using biotinylation followed by incubation with neutravidin. HSP90 is used as loading control (f and g). Full scans of westerns blots are provided in Supplementary Fig. 8. (h) Representative confocal images of mouse peritoneal macrophages from WT and Angptl4−/− mice incubated with Ac-LDL for 24 h and stained with cholera toxin B (CTxB), ABCA1 and DAPI. Quantification of co-localization of CTxB and ABCA1 is on the right panel. Scale bar, 10 µm. (i) Representative images of WT and Angptl4−/− macrophages cultured on coverslips and treated with or without Ac-LDL (120 µg ml−1) in combination with ACAT inhibitor (58035) for 24 h to induce lipid-loading-induced apoptosis (scale bars, 200 µm). Apoptosis was detected using Annexin-V staining. Right panel shows the quantification of percentage of apoptotic cells from four random fields from each cover slip. All data represent the mean±s.e.m. from at least three experiments in duplicate; *P<0.05 compared with WT macrophages by unpaired t-test. MFI, median intensity of fluorescence.

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