ABCA1 Antibody (1276B) - BSA Free NBP2-54792 from Novus Biologicals, a Bio-Techne Brand

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Molecular brain
May 04 2020

Fig 1: AZ7235 increases ABCA1 expression. (a) Cellular ABCA1 protein levels were measured by immunoblot in CCF-STTG1 cells after 72 h treatment with DMSO, 1 µM T0901317, or 3 µM AZ7235. Representative blot was generated from same gel shown in Fig. 1f. (b) CCF-STTG1 cells were labeled with 3H-cholesterol with co-treatment of DMSO alone, 1 µM T0901317, or 3 µM AZ7235 for 24 h. Cholesterol efflux over 24 h in the absence (NA) or presence of 10 µg/ml of lipid-free apoA-I along with the above drug treatment was evaluated. Graphs represent mean % efflux and standard deviation of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 comparing drug effect over respective DMSO control; ### P < 0.001 comparing between NA vs. apoA-I within each drug condition by blocked three-way ANOVA post-hoc tests. (c) Particle size distribution of apoE-containing lipoproteins in the unconcentrated 72-h-conditioned media from drug-treated CCF-STTG1 were assessed by 6% native PAGE followed by immunoblotting for apoE. The ladder on the left represents Stokes diameter. (d-f) Cellular ABCA1 protein levels were measured by immunoblot in primary human astrocytes, HMC3 and primary human brain vascular pericytes after 72 h treatment with vehicle control DMSO, 1 µM T0901317, 3 µM AZ7235. Graphs represent fold-change over DMSO control (dashed line) and +/- 95% confidence intervals from N independent experiments indicated in brackets. *** P < 0.001 compared to vehicle control using blocked two-way ANOVA post-hoc tests. Immunoblot images were cropped to show relevant lanes
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Molecular brain
May 04 2020

Fig 2: AZ7235 has nominal effects on secretion and mRNA levels of apoE and no effect on ABCA1 expression in primary murine mixed glia expressing human apoE3. Primary mixed glial cells (~ 90% astrocytes, 10% microglia) were cultivated from neonatal human APOE3 targeted-replacement mice. Cells were re-plated at 18–20 days in vitro followed by treatment with vehicle (DMSO), 1 µM T0901317, 1 µM and 3 µM AZ7235 for 72 h. (a) Secreted apoE levels in conditioned media were measured by apoE ELISA. The graph represents mean concentration and standard deviation from 4 independent experiments. (b) Representative immunoblot of cellular ABCA1 and apoE with GAPDH as the loading control. Immunoblot images were cropped to show relevant lanes. (c) Quantification of cellular apoE and (d) ABCA1 protein levels in murine glia lysates by immunoblotting. (e-f) Human APOE (e) and mouse Abca1 (f) mRNA levels were measured by qRT-PCR. Graphs represent fold-change over DMSO control (dashed line) and +/- 95% confidence intervals from N independent experiments indicated in brackets. ** P < 0.01, *** P < 0.001 compared to vehicle control by blocked ANOVA post-hoc tests
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Molecular brain
May 04 2020

Fig 3: AZ7235-mediated apoE and ABCA1 induction is LXR-independent. U2-OS LXR-Gal4 chimeric Luciferase reporter assays were used to demonstrate that AZ7235 (1.5 nM – 30 µM) have no direct LXRa (a) or LXRß (b) agonist activity, unlike the direct LXR agonist T091317, after 40 h of treatment. (c) Vehicle control, DMSO, 1 µM T0901317, 3 µM AZ7235 were added to CCF-STTG1 cells transfected with a mixture of LXR-responsive Firefly luciferase construct and constitutively expressing Renilla luciferase construct as an internal control. Luciferase activities were measured after 24 h of treatment. Error bars represent standard deviation from technical triplicates. *** P < 0.0001 by ANOVA post-hoc analysis. (d-e) LXR-knockout (LXRa-/ß-) and LXRa-expressing (LXRa+/ß-) MEF cells were treated with DMSO or drugs for 48 h. Apoe and Abca1 mRNA levels were measured by qRT-PCR. Graph represents fold-change over respective DMSO control (dash line) +/- 95% CI from 5 experiments