The Transcriptional Coactivators p/CIP and SRC-1 Control Insulin Resistance through IRS1 in Obesity Models
Figure 3
Knockdown of p/CIP and SRC-1 in two cell lines leads to increased IRS1 levels and enhanced insulin signaling.
A. Lentiviral shRNA constructs of p/CIP, SRC-1, or both were used to stably knockdown endogenous levels of these coactivators in the F442A fibroblast cell line. 10 μg of total RNAs were used in Northern blot analyses to detect p/CIP and SRC-1 levels. A β-actin probe served as loading control. B. 50 μg total proteins of cell lysates from p/CIP knockdown (PKD), SRC-1 knockdown (SKD), wild-type, and double knockdown (DKD) were resolved by SDS-PAGE gel prior to immunoblot analyses. IRS1 protein levels were significantly increased in the DKD cell line, based on the α-tubulin loading control. C. Immunoblot analyses with stable p/CIP/SRC-1 DKD and IRS1 overexpression F442A cell lines. D. Serum starved F442A cell lines were stimulated for 15 or 30 min with 200 nM insulin, and immunoblot analyses were performed with the cell lysates using the indicated antibodies. Lanes 1, 4, and 7 were wild-type control with a vector construct only, lanes 2, 5, and 8 DKD cell line, and lanes 3, 6, and 9 IRS1 overexpression cell line. E. Immunoblot analyses with C2C12 myoblast stable DKD cell line. Lanes 1, 3, and 5 vector control, and lanes 2, 4, and 6 DKD.