FIG. 4.

Pik3r1 is required for insulin activation of the cdc42/MKK4/JNK pathway. (A) cdc42 activity as determined by PAK1 pull-down assay from liver lysates 3 min after administration of a 5-U portal bolus of insulin (see Materials and Methods). (B) Phosphospecific-antibody blots against phospho-MKK4 and phospho-JNK. The phosphospecific antibody blots were stripped and reprobed with the appropriate antibodies to determine the total levels of the corresponding proteins. (C) Primary hepatocytes isolated from lox/lox or L-Pik3r1KO mice were infected with either LacZ or constitutively active forms of MKK4 and cdc42 (myc tagged), serum starved overnight, and then stimulated with 100 nM insulin. Western blot assays were then performed with the indicated antibodies. (D) Primary hepatocytes of the indicated genotype were infected with either LacZ or a myc-tagged dominant negative cdc42 adenovirus, serum starved overnight, and then stimulated with 100 nM insulin.