Figure 3.

Hepatocyte-specific deletion of GNE promotes the IR degradation in the liver. A–C. Male GNE^fl/fl mice were injected with control AAV8-GFP or AAV8-Cre at 5 weeks of age, and fed control diet for 2 weeks. Mice were fasted for 16 h followed by 4 h refeed, the livers were isolated and the abundance of IRs, EphB4, Clathrin and AP2M1 were assessed by immunoblot after IR immunoprecipitation or in whole liver lysates (A). Quantitation by densitometry in the whole liver lysates (B, N = 6–9/group) or after IR immunoprecipitation (C, N = 8/group). D–I. Male GNE^fl/fl mice were injected with control AAV8-GFP or AAV8-Cre at 5 weeks of age, and fed control diet for 2 weeks, and primary hepatocytes were isolated. The cells were treated with/without 10 nM EphB4 inhibitor NVP (D–F) for 24 h, or with/without 5 mM lysosome inhibitor NH₄Cl for 18 h (G–I). The expression of IRα and IRβ were assessed by immunoblot (N = 6/group). Quantitation of immunoblots by densitometry is shown in E, F, H, and I. Data are represented as mean ± SEM. Significance was determined by unpaired two tailed Student's t test (B and Clathrin in C), unpaired two tailed Student's t test with Welch's correction (EphB4 and Ap2M1 in C) and one-way ANOVA with Tukey's multiple comparison analysis (E–F, H–I).