Analysis of novel insulin-responsive proteins. a, immunoblotting of 14-3-3 pulldowns from L6 myotubes that were either left unstimulated, stimulated with 100 nm insulin for 30 min, or treated with the PI3K inhibitor wortmannin prior to insulin stimulation. b, immunoblotting of 14-3-3 pulldowns from quadriceps of 20-week-old male C57BL/6 mice. Mice were either fasted overnight followed by a mock intraperitoneal injection, ad libitum fed overnight followed by a mock intraperitoneal injection, or fasted overnight followed by an intraperitoneal injection of 1 unit/kg insulin. Mice were sacrificed 10 min after injection, and quadriceps were excised. c, immunoblotting of 14-3-3 pulldowns from L6 myotubes that were stimulated with 100 nm insulin for the indicated times. d, sequence conservation throughout evolution of amino acids surrounding Ser(P)-161 (blue). Critical arginines at −5 and −3 required for AKT phosphorylation are shown in red. e, domain structure of EDC3 showing the location of the putative AKT phosphorylation and 14-3-3 binding site Ser(P)-161. Immunoblots are from a representative experiment (n = 3).