Biological activity of insulin receptor autoantibodies on insulin signaling in vitro. Human hepatic HepG2 cells were incubated with immunoglobulins (Igs) isolated from individual serum samples from InsR-aAb–negative individuals (control) or patients with type B insulin resistance (TBIR). Phosphorylation of the insulin receptor at Tyr1361 (P-Y1361-InsR) was determined by Western blot as a biomarker of insulin signaling. A, The degree of insulin receptor phosphorylation at Y1361 was not different in cells incubated with Ig from control or TBIR serum samples. B) In the presence of insulin (1 ng/mL), insulin receptor phosphorylation at Y1361 was stronger in cells incubated with Ig from control serum in comparison to cells incubated with Ig from InsR-aAb–positive serum samples from patients with TBIR. C, Quantification of the band intensities of P-Y1361-InsR (arrow) by ImageJ analysis supports this notion and highlights the extent of inhibition. The results indicate an antagonistic activity of InsR-aAb in TBIR on insulin-mediated receptor activation.