Ago2 immunoprecipitation (Ago2-IP) experiments after CM or miR-148a transfection were conducted in Huh7 cells as previously described64 (link). Briefly, 1 × 107 cells were transfected with 20 nM miR-148a or CM using RNAimax for 24 h. After 24 h, cells were collected and subjected to Ago2-IP using the RNA isolation kit for human Ago2 (Wako Chemicals) according to the manufacturer’s instructions. The IP pulldown RNA was used to determine the expression levels of miR-148a and LDLR as described above.
In another set of experiments, RISC complexes were immunoprecipitated from the livers of mice that were fasted for 24 h or fasted for 24 h and refed a high-carbohydrate/low fat diet for 12 h using 5 μg of an antibody against mouse Ago2 (2D4) or IgG control as previously described65 (link). Ago2-bound RNA was used to determine the expression levels of miR-148a, ABCA1 and LDLR mRNA as described above. Genes not predicted to be targets of miR-148a (18S, bACTIN and 36B4) were used as negative controls.
In another set of experiments, RISC complexes were immunoprecipitated from the livers of mice that were fasted for 24 h or fasted for 24 h and refed a high-carbohydrate/low fat diet for 12 h using 5 μg of an antibody against mouse Ago2 (2D4) or IgG control as previously described65 (link). Ago2-bound RNA was used to determine the expression levels of miR-148a, ABCA1 and LDLR mRNA as described above. Genes not predicted to be targets of miR-148a (18S, bACTIN and 36B4) were used as negative controls.