Abca1

Protein extraction and Western blot analysis were carried out as described [17 (link)]. Details of the antibody dilutions used for Western blot are as follows: LXRα (1:500 dilution, #ab41902, Abcam, Cambridge, UK), ABCA1 (1:1000, #NB400-105, Novus Biologicals, Cambridge, UK), GAPDH (1:1000, #sc-365062, Santa Cruz Biotechnology, TX, USA), and Calnexin (1:1000, #sc-11397, Santa Cruz Biotechnology, TX, USA).
RNA extraction and quantitative real time PCR are described in detail in the Supplementary material. In short, total RNA was extracted with Trifast (Peqlab, Erlangen, Germany) and reverse transcribed (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Carlsbad, CA). The quantity of RNA used in quantitative real time PCR was optimized prior to obtain ct values within the range of 18–30.
For mature miR-206 quantification the miRCURY LNA™ Universal RT microRNA PCR system (Exiqon, Vedbaek, Denmark) was used together with miR-206 or U6 (reference gene) specific predesigned LNA primers. Quantitative real time PCR was carried out using gene specific primers and QuantiFast SYBR Green PCR Kit (Qiagen, Germany) on LC480 (Roche Diagnostics, Basel, Switzerland). All samples were normalized to cyclophilin A mRNA expression. Experiments were carried out in triplicates.

Total cell lysates were prepared in RIPA buffer supplemented with protease inhibitors (Roche Molecular Biochemicals) and PMSF. Samples (10–40 μg) were separated on NuPAGE Bis-Tris gels (Invitrogen) and transferred to nitrocellulose. Membranes were probed with the following antibodies: LDLR (1:1000; Cayman Chemical or AbCam), ABCA1 (1:1000; Novus), actin (1:10,000; Sigma), and tubulin (1:3000 Calbiochem). Horseradish peroxidase (HRP)–conjugated secondary antibodies (Zymed) were used, and antibody binding was visualized by chemiluminescence (ECL Plus GE Healthcare). Blots were quantified by densitometry with ImageJ software (version 1.42q; National Institutes of Health).