FIGURE 2.

IR mutations prevent ADDL degradation. The human full-length wild-type IR (hIR-wt), or two IR mutations (IR-1153IIe and IR1030Ala) were transient transfected to primary hippocampal neurons or NIH3T3 cells. Cells were then treated with biotin-labeled ADDLs (bADDLs) in the presence or absence of insulin. The media were collected and concentrated. The cell lysates were prepared with a lysates buffer. Both the extracellular and the cell-attached bADDLs were measured with dot blots using NU1. A, the insulin-stimulated IR tyrosine phosphorylation from NIH3T3 cells transfected with huIR-wt and mutated human IR. Only the huIR-wt showed positive insulin-induced tyrosine phosphorylation on the β-subunit. B, bADDL digestions in the medium were inhibited in the IR mutants-transfected NIH 3T3 cells. C-1, bADDL digestions in the medium were inhibited in the IR mutants-transfected hippocampal neurons. C-2, insulin-induced translocation of ADDLs were not seen in the IR mutant-transfected hippocampal cells. **, p < 0.001, n = 3. C-3, transfected neurons were treated with bADDLs. The concentrated medium from different conditions was pulled down by streptavidin covalently immobilized to agarose resin and detected on Western blots with 6E10. bADDL species were increased in the medium of primary neurons transfected with IR mutants.