rabbit polyclonal antibody against abca1  (Novus Biologicals)

Journal: Biomedicines

Article Title: Small GTPase ARL4C Associated with Various Cancers Affects Microtubule Nucleation.

doi: 10.3390/biomedicines12122872

Figure Lengend Snippet: Figure 2. Treatment using LXR/RXR ligands does not lead to an increase in the endogenous ABCA1 or ARL4C level in many cell lines: (A) Titration of BS-C-1 lysate demonstrates that the ratio of levels of ARL4C in Vero and BS-C-1 cells is 1:4.5. (B) The level of ARL4C is not increased in both Vero and BS-C-1 cells even after 7 days of treatment with T0901317 and bexarotene. (C) LXR/RXR-dependent pathway is activated in HeLa cells and weakly activated in MCF7 cells after 48h exposure to 2.5 µM T0901317/bexarotene; however, not in Vero cells even after prolonged treatment. (D) Neither ABCA1 nor ARL4C levels increase after treatment of COS7 and U2OS cells with RXR/LXR ligands. Long-term treatment of the MCF7 cells results in only a slight increase in the ARL4C level.

Article Snippet: Then, the membrane was blocked with 5% skimmed milk in TBST for 1 h. Rabbit polyclonal antibody against ARL4C (Novus Biologicals, Centennial, CO, USA), mouse monoclonal antibody against GAPDH (ThermoFisher Scientific, Waltham, MA, USA), rabbit polyclonal antibody against ABCA1 (Novus Biologicals, Centennial, CO, USA), mouse monoclonal antibody against α-tubulin, and clone DM1A (Santa Cruz Biotechnology, Dallas, TX, USA) were used as primary antibodies.

Techniques: Titration

Journal: Chinese Medicine

Article Title: Combination of mangiferin and T0901317 targeting autophagy promotes cholesterol efflux from macrophage foam cell in atherosclerosis

doi: 10.1186/s13020-023-00876-9

Figure Lengend Snippet: Treatment of T0 and MGF activates expression of LXR target molecules in macrophage and inhibits foam cell formation. A Peritoneal macrophages collected from mice were stained with Oil Red O or anti-bodipy antibody (green) to evaluate formation of foam cells (> 10 lipid droplets per cell, > 10 fields per sample), scale bar, 20 μm; and cholesterol contents were measured. *** p < 0.001, n = 3. Expression of ( B ) ABCA1 and ( C ) ABCG1 (stained with red fluorescent color) was determined by immunofluorescent staining of aortic root cross sections from Apoe −/− mice and quantified by a computer-assisted image analysis protocol. Scale bar, 100 μm. ** p < 0.01, n = 3. Expression of ABCA1, ABCG1, LXRα and CD36 in cultured peritoneal macrophages ( D ) and RAW264.7 cells ( E ) was determined by western blot with total proteins extracted from cell samples. *** p < 0.001, ** p < 0.01, * p < 0.05 (n = 3)

Article Snippet: Primary rabbit polyclonal antibodies against ABCA1 (ab18180), ABCG1 (ab218528), LC3 (ab192890), SREBP-1c (ab28481), FAS (ab133619), and β-actin (ab8227) were purchased from Abcam (Cambridge, MA, USA).

Techniques: Expressing, Staining, Cell Culture, Western Blot

Journal: Chinese Medicine

Article Title: Combination of mangiferin and T0901317 targeting autophagy promotes cholesterol efflux from macrophage foam cell in atherosclerosis

doi: 10.1186/s13020-023-00876-9

Figure Lengend Snippet: Model for the function of combined treatment of T0 and MGF in the AS related dyslipidemia targeting autophagy in cholesterol efflux from macrophage foam cells. On the one hand, T0 alone or T0 and MGF promote macrophage cholesterol efflux by activating LXRα to augment the expression of ABCA1 and ABCG1 and also enhance lipophagy in atherosclerotic lesion. Of note, in mammalian macrophage foam cells, lipid droplets are tagged for autophagic fusion, possibly beginning with mTOR-AMPK signaling to initiate lipid droplet degradation for further cholesterol depletion. On the other hand, T0-induced hepatic lipid abnormal accumulation is attenuated by MGF. In this scenario, MGF may activate AMPK signaling to suppress lipid synthesis and accelerate lipolysis for β-oxidation of free fatty acid. Alternatively, hepatic lipid droplets may be degraded through autophagy mechanism mediated by AMPK, and relative classical factors LC3, ATGs and p38 to facilitate hepatic lipophagy

Article Snippet: Primary rabbit polyclonal antibodies against ABCA1 (ab18180), ABCG1 (ab218528), LC3 (ab192890), SREBP-1c (ab28481), FAS (ab133619), and β-actin (ab8227) were purchased from Abcam (Cambridge, MA, USA).

Techniques: Expressing

Journal: Nutrients

Article Title: Effects of 2-Methoxyestradiol, a Main Metabolite of Estradiol on Hepatic ABCA1 Expression in HepG2 Cells

doi: 10.3390/nu14020288

Figure Lengend Snippet: Effects of 2-ME 2 on the expression of ABCA1 in HepG2 cells. ( A ) ABCA1 protein expression in HepG2 cells treated with 2-ME 2 at varying doses for 24 h. ( B ) Effect of 2-ME 2 on the mRNA level of ABCA1 expression. An abundance of GAPDH served as the control, and the relative expression of ABCA1 to GAPDH is shown as a percent of the control in the histogram. The histogram shows the mean ± SEM ( n = 3) of separate experiments for each group. * p < 0.05 compared to 0.

Article Snippet: The membranes with proteins were blocked with skim milk overnight and then incubated with 3% BSA diluted in PBS with 0.1% Tween 20 (PBS-T) containing rabbit polyclonal antibody, the first antibody against ABCA1 (1:500; Santa Cruz, sc-20794), Akt (Upstate, 28740; 1:1000), FoxO1 (1:1000; CST, 2880s), p-Akt Ser 473 (CST, 9271s; 1:1000), p-FoxO1 Ser256 (1:1000; CST, 9461s), GAPDH (1:5000; TREVIGEN, 2275-PC-100), or nuclear TFIID (1:200; Santa Cruz, sc-273) overnight and incubated with horseradish peroxidase (HRP)-linked antirabbit IgG secondary antibody (1:2000; DakoCytomation, P0448) for 1 h at 4 °C.

Techniques: Expressing, Control

Journal: Nutrients

Article Title: Effects of 2-Methoxyestradiol, a Main Metabolite of Estradiol on Hepatic ABCA1 Expression in HepG2 Cells

doi: 10.3390/nu14020288

Figure Lengend Snippet: 2-ME 2 enhances the promoter activity of ABCA1 via the PI3K/Akt signaling pathway. ( A ) ABCA1 promoter activity in HepG2 cells treated with 2-ME 2 at varying doses for 24 h. ( B ) Effects of a PI3K inhibitor LY294002 (LY), an MEK/ERK inhibitor PD98059 (PD), or a PKC inhibitor bisindolylmaleimide I (BIS) on 2-ME 2 -induced ABCA1 promoter activity in HepG2 cells. ( C ) Protein expression of ABCA1 in HepG2 cells treated with 2-ME 2 with or without LY, PD, or BIS. ( D ) Role of the PI3K/Akt pathway on ABCA1 promoter activity induced by 2-ME 2 . pcDNA, control vector; p110, overexpression of the p110 catalytic subunit of PI3K; Akt, overexpression of Akt; Akt-DN, domain-negative Akt. Each data shows the mean ± SEM ( n = 3) of separate experiments for each group. * p < 0.05 compared to control or pcDNA; # p < 0.05 compared to 2-ME 2 .

Article Snippet: The membranes with proteins were blocked with skim milk overnight and then incubated with 3% BSA diluted in PBS with 0.1% Tween 20 (PBS-T) containing rabbit polyclonal antibody, the first antibody against ABCA1 (1:500; Santa Cruz, sc-20794), Akt (Upstate, 28740; 1:1000), FoxO1 (1:1000; CST, 2880s), p-Akt Ser 473 (CST, 9271s; 1:1000), p-FoxO1 Ser256 (1:1000; CST, 9461s), GAPDH (1:5000; TREVIGEN, 2275-PC-100), or nuclear TFIID (1:200; Santa Cruz, sc-273) overnight and incubated with horseradish peroxidase (HRP)-linked antirabbit IgG secondary antibody (1:2000; DakoCytomation, P0448) for 1 h at 4 °C.

Techniques: Activity Assay, Expressing, Control, Plasmid Preparation, Over Expression

Journal: Nutrients

Article Title: Effects of 2-Methoxyestradiol, a Main Metabolite of Estradiol on Hepatic ABCA1 Expression in HepG2 Cells

doi: 10.3390/nu14020288

Figure Lengend Snippet: Role of FoxO1 in 2-ME 2 -mediated ABCA1 transcription in HepG2 cells. ( A ) Effect of FoxO1 on ABCA1 promoter activity. ( B ) Binding of FoxO1 to the ABCA1 promoter region. FoxO1 specifically immunoprecipitates ABCA1 chromatin by the ChIP assay. No ChIP was detected when the chromatin was immunoprecipitated with an unspecific negative control IgG (IgG). ( C ) ChIP-real time PCR showed that 2-ME 2 reduced the binding of FoxO1 to ABCA1 promoter. ( D ), Effect of 2-ME 2 on FoxO1-binding sites-mutated promoter activity (5′-AACA-3′ to 5′-GGAG-3′). ( E ) Effect of FoxO1, Akt, or p110 on FoxO1-binding sites-mutated promoter activity. ( F ) Expression of nuclear FoxO1 after silencing FoxO1 with specific siRNA. ( G , H ) Effect of 2-ME 2 on the protein ( G ) and mRNA ( H ) expression of ABCA1 after silencing FoxO1. siCon, scramble siRNA; siFoxO1, FoxO1-specific siRNA. Each data point shows the mean ± SEM ( n = 3) of separate experiments for each group. * p < 0.05 compared to 0; N.S., no significant difference.

Article Snippet: The membranes with proteins were blocked with skim milk overnight and then incubated with 3% BSA diluted in PBS with 0.1% Tween 20 (PBS-T) containing rabbit polyclonal antibody, the first antibody against ABCA1 (1:500; Santa Cruz, sc-20794), Akt (Upstate, 28740; 1:1000), FoxO1 (1:1000; CST, 2880s), p-Akt Ser 473 (CST, 9271s; 1:1000), p-FoxO1 Ser256 (1:1000; CST, 9461s), GAPDH (1:5000; TREVIGEN, 2275-PC-100), or nuclear TFIID (1:200; Santa Cruz, sc-273) overnight and incubated with horseradish peroxidase (HRP)-linked antirabbit IgG secondary antibody (1:2000; DakoCytomation, P0448) for 1 h at 4 °C.

Techniques: Activity Assay, Binding Assay, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Expressing

Journal: Nutrients

Article Title: Effects of 2-Methoxyestradiol, a Main Metabolite of Estradiol on Hepatic ABCA1 Expression in HepG2 Cells

doi: 10.3390/nu14020288

Figure Lengend Snippet: Schematic diagram of the mechanisms in 2-ME 2 -suppressed cholesterol accumulation by the upregulation of ABCA1 in HepG2 cells.

Article Snippet: The membranes with proteins were blocked with skim milk overnight and then incubated with 3% BSA diluted in PBS with 0.1% Tween 20 (PBS-T) containing rabbit polyclonal antibody, the first antibody against ABCA1 (1:500; Santa Cruz, sc-20794), Akt (Upstate, 28740; 1:1000), FoxO1 (1:1000; CST, 2880s), p-Akt Ser 473 (CST, 9271s; 1:1000), p-FoxO1 Ser256 (1:1000; CST, 9461s), GAPDH (1:5000; TREVIGEN, 2275-PC-100), or nuclear TFIID (1:200; Santa Cruz, sc-273) overnight and incubated with horseradish peroxidase (HRP)-linked antirabbit IgG secondary antibody (1:2000; DakoCytomation, P0448) for 1 h at 4 °C.

Techniques:

Journal: Cell Death & Disease

Article Title: CTRP12 ameliorates atherosclerosis by promoting cholesterol efflux and inhibiting inflammatory response via the miR-155-5p/LXRα pathway

doi: 10.1038/s41419-021-03544-8

Figure Lengend Snippet: A – D THP-1 macrophages were incubated with 50 µg/mL ox-LDL for 48 h, followed by treatment with PBS, LV-NC, or LV-CTRP12 for 72 h. A , B The expression of ABCA1 and ABCG1 was determined by qRT-PCR and western blot. C , D Detection of CD36 and SR-A expression using qRT-PCR and western blot. Data are expressed as the mean ± SD from three independent experiments. **** P < 0.0001; ns not significant.

Article Snippet: Then, proteins were subjected to SDS-PAGE, followed by immunoblotting with rabbit polyclonal antibody against CTRP12 (PA5-46452, 1:500, ThermoFisher), rabbit polyclonal antibody against ABCA1 (PA1-16789, 1:800, ThermoFisher), rabbit polyclonal antibody against ABCG1 (GTX30598, 1:500, GeneTex, Irvine, CA, USA), rabbit polyclonal antibody against CD36 (PA1-16813, 1:1000, ThermoFisher), mouse monoclonal antibody against SR-A (sc-166184, 1:500, Santa Cruz, TX, USA), rabbit polyclonal antibody against LXRα (L5044, 1:1000, Sigma-Aldrich) and rabbit monoclonal antibody against β-actin (ab115777, 1:1000, Abcam, Cambridge, MA, USA).

Techniques: Incubation, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: CTRP12 ameliorates atherosclerosis by promoting cholesterol efflux and inhibiting inflammatory response via the miR-155-5p/LXRα pathway

doi: 10.1038/s41419-021-03544-8

Figure Lengend Snippet: A THP-1 macrophages were treated with 50 µg/mL ox-LDL for 48 h, followed by transfection with PBS, LV-NC, or LV-CTRP12 for 72 h. LXRα expression was determined by qRT-PCR and western blot. B After transfection with scrambled siRNA or LXRα siRNA for 48 h, cell lysates were immunoblotted with indicated antibodies. C – E THP-1 macrophages were loaded with 50 µg/mL ox-LDL for 48 h, transfected with LXRα siRNA for another 48 h and then treated with LV-CTRP12 for 72 h. C The mRNA and protein levels of ABCA1 and ABCG1 were detected by qRT-PCR and western blot, respectively. D , E Representative fluorescent images of NBD-cholesterol burden (×200) and quantitative analyses of cholesterol efflux to apoA-I and HDL. Scale bar = 20 μm. Data are expressed as the mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Then, proteins were subjected to SDS-PAGE, followed by immunoblotting with rabbit polyclonal antibody against CTRP12 (PA5-46452, 1:500, ThermoFisher), rabbit polyclonal antibody against ABCA1 (PA1-16789, 1:800, ThermoFisher), rabbit polyclonal antibody against ABCG1 (GTX30598, 1:500, GeneTex, Irvine, CA, USA), rabbit polyclonal antibody against CD36 (PA1-16813, 1:1000, ThermoFisher), mouse monoclonal antibody against SR-A (sc-166184, 1:500, Santa Cruz, TX, USA), rabbit polyclonal antibody against LXRα (L5044, 1:1000, Sigma-Aldrich) and rabbit monoclonal antibody against β-actin (ab115777, 1:1000, Abcam, Cambridge, MA, USA).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: CTRP12 ameliorates atherosclerosis by promoting cholesterol efflux and inhibiting inflammatory response via the miR-155-5p/LXRα pathway

doi: 10.1038/s41419-021-03544-8

Figure Lengend Snippet: A THP-1 macrophages were treated with 50 µg/mL ox-LDL for 48 h and then transfected with PBS, LV-NC, or LV-CTRP12 for 72 h, followed by detection of miR-155-5p expression using qRT-PCR. B – D THP-1 macrophages were loaded with 50 µg/mL ox-LDL for 48 h, transfected with miR-155-5p mimic for another 48 h and then transduced with LV-CTRP12 for 72 h. The mRNA and protein levels of LXRα, ABCA1, and ABCG1 were detected by qRT-PCR and western blot, respectively. Data are represented as the mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Then, proteins were subjected to SDS-PAGE, followed by immunoblotting with rabbit polyclonal antibody against CTRP12 (PA5-46452, 1:500, ThermoFisher), rabbit polyclonal antibody against ABCA1 (PA1-16789, 1:800, ThermoFisher), rabbit polyclonal antibody against ABCG1 (GTX30598, 1:500, GeneTex, Irvine, CA, USA), rabbit polyclonal antibody against CD36 (PA1-16813, 1:1000, ThermoFisher), mouse monoclonal antibody against SR-A (sc-166184, 1:500, Santa Cruz, TX, USA), rabbit polyclonal antibody against LXRα (L5044, 1:1000, Sigma-Aldrich) and rabbit monoclonal antibody against β-actin (ab115777, 1:1000, Abcam, Cambridge, MA, USA).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Transduction, Western Blot

Journal: Cell Death & Disease

Article Title: CTRP12 ameliorates atherosclerosis by promoting cholesterol efflux and inhibiting inflammatory response via the miR-155-5p/LXRα pathway

doi: 10.1038/s41419-021-03544-8

Figure Lengend Snippet: A Plasma levels of TC, TG, HDL-C, and LDL-C were determined using the commercial kits ( n = 10). B Mice were injected intraperitoneally with [ 3 H]-cholesterol-labeled J774 macrophages. The radioactivity in the plasma, liver, and feces were assessed by a liquid scintillation counter ( n = 5). C , D Detection of miR-155-5p, LXRα, ABCA1, and ABCG1 expression in the aortas by qRT-PCR and western blot ( n = 10). Data are represented as the mean ± SD. * P < 0.05, **** P < 0.0001; ns not significant.

Article Snippet: Then, proteins were subjected to SDS-PAGE, followed by immunoblotting with rabbit polyclonal antibody against CTRP12 (PA5-46452, 1:500, ThermoFisher), rabbit polyclonal antibody against ABCA1 (PA1-16789, 1:800, ThermoFisher), rabbit polyclonal antibody against ABCG1 (GTX30598, 1:500, GeneTex, Irvine, CA, USA), rabbit polyclonal antibody against CD36 (PA1-16813, 1:1000, ThermoFisher), mouse monoclonal antibody against SR-A (sc-166184, 1:500, Santa Cruz, TX, USA), rabbit polyclonal antibody against LXRα (L5044, 1:1000, Sigma-Aldrich) and rabbit monoclonal antibody against β-actin (ab115777, 1:1000, Abcam, Cambridge, MA, USA).

Techniques: Injection, Labeling, Radioactivity, Expressing, Quantitative RT-PCR, Western Blot