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. Author manuscript; available in PMC: 2014 Jul 2.
Published in final edited form as: Cell Metab. 2013 Jul 2;18(1):51–61. doi: 10.1016/j.cmet.2013.06.010

Figure 5. Regulation of SREBPs by the miR-182 locus through FBXW7 is conserved in human cells.

Figure 5

A) HeLa cells were transfected with siRNA or pre-miRs as indicated (10 nM, Ambion) in antibiotic-free medium as described in Materials and Methods. After 24 hours, the dishes were switched to DMEM containing 5% lipoprotein-deficient serum, 12 µg/mL cholesterol, 1 µg/mL 25-HC and incubated for 24 hours at 37°C. (A) qPCR for FBXW7 and immunoblotting for FBXW7, SREBP-1 and β-actin. B) The full length FBXW7 coding sequence was cloned downstream of the constitutive RPL10 promoter with that natural FBXW7 3’UTR intact. We also prepared a version where the two predicted miR-182 targeting sites were mutated to decrease the predicted complementarity. These constructs were transfected into HeLa cells in combination with miR-182 or controls and cultured as described in Methods and in the Figure diagram. Quantitation from a scanned image of the immunoblot is presented at the top. Data are represented as mean +/− SD. See also Figure S6.