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. 2023 Jul 8;75:101767. doi: 10.1016/j.molmet.2023.101767

Figure 6.

Figure 6

Ube4A mediates insulin signaling via K63-Ub of Akt and APPL1.

A-B. Stimulatory phosphorylation (S473) of Akt in the liver of HFD-fed mice (N = 4/cohort).

C-E. Acute insulin-induced stimulatory phosphorylation of Akt (S473 and T308) in the liver of young, chow-fed mice. Quantification of 3 independent experiments is shown.

F-G. Acute insulin-induced stimulatory phosphorylation of Akt (S473 and T308) in hepatocytes in vitro. Quantification of 3 independent experiments is shown.

H–I. Acute insulin-induced stimulatory phosphorylation of Akt (S473 and T308) in primary fibroblasts in vitro. Quantification of 3 independent experiments is shown.

J-K. Effects of adenovirus (AV)-mediated overexpression of Ube4A on acute insulin-induced stimulatory phosphorylation of Akt (S473 and T308) in primary fibroblasts and hepatocytes. In fibroblasts, the Akt-T308 antibody detected a non-specific band at a lower molecular weight, which did not respond to insulin.

L-M. K63-Ub of Akt in the liver of untreated and acute insulin-treated young, chow-fed mice. Data represents 3 independent experiments.

N. Ube4A (FL)-mediated K63-Ub of Akt in vitro. Ube4A-ΔU-box was used as a negative control. Data represents at least 3 independent experiments.

O. Top panel: LC-MS/MS-based identification of the exclusive unique spectrum counts of immunoprecipitated Ube4A and co-immunoprecipitated APPL1 from the BAT of young, chow-fed WT mice. Ig-control samples did not show any spectrum. Bottom panel: LC-MS/MS-based identification of the APPL1 peptides in the above experiment.

P-Q. Acute insulin-induced K63-Ub of APPL1 and APPL1-Akt binding in the liver of UKO mice. K63-Ub was detected in immunoprecipitated APPL1. Co-precipitated Akt was detected by immunoblotting in APPL1-immunoprecipitated samples. Data represents 3 independent experiments.

Each figure represents the conclusion from at least three independent experiments. Mean ± s.e.m. shown within dot plots. For multiple comparisons, two-way ANOVA with Holm-Šidák multiple comparison test and for 2 independent data sets, Two-tailed unpaired Student's t-test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.