Figure 2

tyrHDL increases ABCA1 protein but not mRNA levels. Fibroblasts grown to confluence, cholesterol loaded, and equilibrated as in Figure 1 were treated with medium containing 1 mg/ml BSA alone or plus 10 μg/ml apoA-I, HDL or tyrHDL for 2 or 16 h. (A) mRNA was collected using Trizol™ extraction at the indicated time and qPCR was performed with cyclophilin used as the internal control. Data represent Avg ± SD for triplicates of each sample and are representative of three experiments with similar results. *, p < 0.01 when compared with BSA-treated cells at 16 h. (B) Cells were collected in N-dodecyl-β-D-maltoside lysis buffer with proteinase inhibitors, and ABCA1 protein was determined following separation of proteins on 5-15% gradient gels and immunoblotting. Values at the top of lanes represent the ratio of densitometer readings of ABCA1 protein normalized for protein disulfide isomerase (PDI) loading control, with this ratio in cholesterol-loaded cells at 0 h set as 1. The data are representative of three experiments with similar results. Chol- indicates cells not loaded with cholesterol loading prior to analyses.