A. 2.5μg of plasmid encoding Flag-tagged JNK1 was transfected into NIH3T3 L1 cells along with 2.5μg of GPS1, 2.5μg Asb-4, or both. The amount of plasmid DNA in each transfection was equalized with pCMV empty vector. JNK was immunoprecipitated with anti-FLAG M2 antibody-conjugated agarose and JNK activity was assayed using an immunocomplex kinase assay with c-Jun (2μg each sample) as substrate. Column 2 vs 1, +p < 0.05; Column 3 vs 2, **P < 0.01. B. JNK activity was assayed in the presence of insulin (20 nM). Column 2 vs 1, #p < 0.05; Column 3 vs 2, +p < 0.01. Column 4 vs 3, **p < 0.01. C. 2.5μg of plasmid encoding HA-tagged IRS-1 was transfected into HEK293 cells along with 2.5μg of GPS1, 2.5μg Asb-4, or both. The cell lysates were blotted with anti-phospho-IRS-1ser307 antibody. Column 2 vs 1, +p < 0.05; Column 3 vs 2, **P < 0.01.