Insulin signaling was enhanced in DEP-1 ASO mice. A: Immunoblotting analysis of liver lysates in control ASO or DEP-1 ASO treated mice challenged with intraveneous injection of insulin (10 U/kg body weight, 2 min) prior to euthanasia; (n = 3 per group). Key intermediates of the insulin signaling cascade were analyzed with indicated antibodies. GAPDH was used as the loading control. B-D: Densitometric analysis of immunoblotting for pTyr 99 (normalization to IR), pAkt (Ser 473) and pAkt (Thr 308) (normalization to Akt) were performed and expressed as arbitrary units; (n = 3 per group). *P < 0.05, **P < 0.01. E: Detection of phosphorylated insulin receptor in liver tissue of mice. Insulin receptor tyrosine phosphorylation was detected by in situ PLA (red dots) applying anti-insulin receptor and anti-phosphotyrosine antibodies. Liver tissue sections derived from C57BL/6J mice subjected to either control ASOs or DEP-1 ASOs and insulin stimulation, as described in A. The cells were counterstained with DAPI (blue) to visualize the nuclei. Specificity of antibody binding and PLA method is demonstrated by control sections with either only antibodies against the insulin receptor (IR), against phosphotyrosine (pY100), or omitting primary antibodies (none). Insets of representative cells are shown. Scale bars represent 25 μm. F: The quantification of PLA signals per cell for animal groups is depicted as the percentage of each population with a certain number of RCPs per cell (n = 424–458 cells per group).