Detection of the recruitment of DEP-1 to the insulin receptor. A: The recruitment of DEP-1 to the insulin receptor was detected by using in situ PLA (red dots) in liver tissue sections of C57BL/6J mice which were either untreated or stimulated with insulin (10 U/kg body weight, 2 min). The cells were counterstained with DAPI (blue) to visualize the nuclei. Insets of representative cells are shown. Scale bars represent 25 μm; none, no primary antibody; insulin receptor (IR), rabbit anti-IR antibody; DEP-1, goat anti-DEP-1 antibody. B: The quantification of PLA signals per cell for both groups is depicted as the percentage of each population with a certain number of RCPs per cell (n = 350–500 cells). C: AML12 liver cells were used for evaluation of insulin receptor dephosphorylation by PTPs. Starved cells were left resting (−) or were stimulated with 100 nM insulin (+) for 10 min, followed by cell lysis and protein isolation. The immunoprecipitated insulin receptor was subjected to recombinant DEP-1 or PTP1B, as indicated, with or without preincubation with sodium vanadate (n = 3 experiments). Immunoblotting was performed with depicted antibodies.