FIGURE 4.

CIP4 negatively regulates surface GLUT4 expression through Akt-independent signaling. A, subcellular fractions were isolated from whole tissue lysate of mouse skeletal muscle (quadriceps femoralis) collected from the top of the gradient (fraction 1) to the bottom of the gradient (fraction 9), with the most buoyant fractions (fractions 1, 2, and 3) representing plasma membrane. P1 and P2 contain almost all of the plasma membrane and T tubule marker proteins (20). P1 contains most of the nuclei, mitochondria, and other heavy organelles. P1 also contains surface membrane. GLUT4 also resides within the intracellular membrane fractions. Protein concentration was normalized for each fractionation. B, wild-type or homozygous Cip4-null mice were fasted overnight followed by intraperitoneal injection of insulin. 20 min after injection, muscle and heart tissues were harvested and lysates prepared. The lysates were used for Western blotting for pAkt and Akt. wt, wild-type; ko, knock-out.