Rapid degradation of ABCA1 protein following cAMP withdrawal and treatment with PKA inhibitor suggests ABCA1 is a short-lived protein primarily regulated at the transcriptional level

摘要

Panel A, Effect of serum on 8-Br-cAMP-induced regulation of ABCA1. The transfected HEK293 cell line was incubated in the absence or presence of serum with or without 8-Br-cAMP. 8-Br-cAMP treatment was done for 24 h. Cell lysate (25 μg protein) was separated on 8–16% Tris- Glycine gel and transferred to nitrocellulose membrane followed by detection with either ABCA1 antibody or a polyclonal anti-beta actin antibody. The ABCA1 and β-actin proteins are shown by arrows. Panel B, Effect of cAMP on cholesterol efflux in WT 293 cells, and in transfected 293 cells in the presence and absence of apoAI and 8-Br-cAMP. The details of the efflux assay are provided in the methods section. Dotted bars, 293 cells, shaded bars, 293 cells with 8-Br-cAMP, big dotted bars, transfected 293 cells, filled bars, transfected 293 cells with 8-Br-cAMP. Panel C, Effect of 8-Br-cAMP withdrawal on ABCA1 protein levels in the 293 cells stably transfected with hABCA1. The transfected cells were treated with or without cAMP for 24 h, the medium was removed, and cells were further incubated in the same media but without cAMP for 0 h, 2 h, 6 h, and 24 h, as indicated after the + sign. The cell lysates were prepared and ABCA1 and β-actin proteins were measured by Western blotting. The respective protein bands of ABCA1 and β-actin are indicated by arrows. Panel D, Effect of 8-Br-cAMP withdrawal on ABCA1 mRNA levels in the 293 cells stably transfected with hABCA1. Cells were treated with or without 8-Br-cAMP as described in Panel C, and RNA was prepared from the cells. Sixty nanogram of the total RNA were taken for ABCA1 mRNA quantitation by real-time RT-PCR using Taqman 7900HT. As a control 18S RNA was also quantitated. The numbers after the + sign indicates time in hour after cAMP treatment. cAMP- indicates without cAMP, and cAMP + indicates with cAMP