Fig 2.

CaMKK cascade stimulates the ABCA1 promoter activity in response to exendin-4. (A) Phosphorylation of CaMKIV by exendin-4 in INS-1 cells. INS-1 cells were exposed to 10 nM exendin-4 for 2 min. before harvest at the predetermined time intervals. The total cell extracts were subjected to immunoprecipitation using anti-CaMKIV antibodies and SDS-PAGE, followed by Western blotting analysis using anti-phospho-Thr196 antibodies (upper insert). Total cell lysates were also blotted using anti-CaMKIV antibodies as a control (lower insert). (B) Effect of CaMK cascade on ABCA1 promoter activity. Cells were transfected with pABCA1-LUC and an empty vector, or CaMKIVc expression vectors. The cells were incubated for 24 hrs after transfection. Each data point shows the mean ± S.E. of three separate transfections that were performed on separate days. The asterisk denotes a significant difference (P < 0.01). (C) Effects of CaMKIV knockdown on ABCA1 expression in INS-1 cells. SiRNA of CaMKIV (siCaMKIV) or scrambled siRNA (siCont) was transfected into INS-1 cells, and then treated with exendin-4 (Ex-4). At 24 hrs after transfection, the abundance of ABCA1 protein level was measured using Western blot analysis (upper panel). The ratio of ABCA1 to GAPDH is shown as the percentage of control. Each data point shows the mean ± S.E. (n= 3) of separate experiments. The asterisk denotes a significant difference (P < 0.05). N.S.; no significant difference.