Alpha word for mac11/8/2022 All the transferred membranes were blocked in 5% milk in TBST (TBS, 0.1% v/ v Tween 20) for at least 1 h. For detecting ABCA1 proteins, the protein samples were incubated at room temperature for 20 min, loaded into a 4.8% SDS-PAGE gel, run overnight at 4 ☌, and transferred onto Immobilon-P PVDF Membrane (Millipore Sigma, Burlington, MA, USA). For detecting ABCG1 and alpha-tubulin, the samples were loaded into an 8% SDS-PAGE gel and transferred using the semi-dry transfer method (Bio-Rad Trans Blot Turbo Transfer System). The protein concentration was normalized with 6X Laemmli SDS sample buffer (Alfa Aesar, Tewksbury, MA, USA) and water and heated at 95 ☌ for 10 min. Supernatant was collected, and protein concentration was measured using Pierce BCA protein assay (Thermo Fisher Scientific, Rockford, IL, USA). Lysates were rotated at 4 ☌ and then centrifuged at 15,000× g rpm for 30 min. Western BlottingĬells were collected into ice-cold protein lysis buffer containing protease inhibitors after washing with sterile PBS. The primer sequences for qRT-PCR are presented in Table S1 in Supplementary Information. Relative mRNA levels were quantified using the 2 −ΔΔCT method and normalized to 36b4. Conditions for the qPCR reaction were as follows: 50 ☌ for 2 min, 95 ☌ for 10 min, 40 cycles of 95 ☌ for 15 s, and 60 ☌ for 1 min. The cDNA was diluted further, and mRNA levels were quantified by quantitative real-time PCR (qRT-PCR) using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Austin, TX, USA) on a 7500 Real Time PCR machine (Applied Biosystems, Foster City, CA, USA). Briefly, samples were incubated at 25 ☌ for 10 min, 48 ☌ for 30 min, and then 95 ☌ for 5 min using a thermal cycler program. The cDNA synthesis was performed using the TaqMan Reverse Transcription Kit (Thermo Fisher Scientific, Carlsbad, CA, USA) from total RNA according to the manufacturer’s instructions. After washing with 70% ethanol, RNA pellet was air dried for 5–10 min and then dissolved in nuclease-free water. The supernatant was collected, and equal volume of isopropanol was added and centrifuged at 12,000× g rpm for 20 min to the precipitate RNA. Chloroform was added, followed by vortex for 10–15 s and centrifugation at 12,000× g rpm for 15 min. About 100 mg tissues were homogenized in TRIzol reagent. Our data demonstrate that macrophage RARα protects against atherosclerosis, likely via inducing cholesterol efflux and inhibiting inflammation.Ĭells were collected in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) after washing with PBS. In Ldlr -/- mice, myeloid ablation of RARα significantly reduced macrophage Abca1 and Abcg1 expression and cholesterol efflux, induced inflammatory genes, and aggravated Western diet-induced atherosclerosis. All-trans retinoic acid (AtRA) induced ATP-binding cassette subfamily A member 1 ( Abca1) and Abcg1 expression and cholesterol efflux in both Rarα Mac-/- mice and Rarα fl/fl mice. Macrophages isolated from myeloid-specific Rarα -/- ( Rarα Mac-/-) mice showed increased lipid accumulation and inflammation and reduced cholesterol efflux compared to Rarα fl/fl (control) mice. In the current study, we investigated the role of macrophage RARα in the development of atherosclerosis. However, the role of retinoic acid receptor alpha (RARα) in atherosclerosis remains to be determined. Retinoic acid signaling plays an important role in regulating lipid metabolism and inflammation.
0 Comments
Leave a Reply.AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |