Fig. 5: Inactivation of Rhbdf2–MAP3K7 axis is essential for Trim31 function.

a Representative immunofluorescence images of Rhbdf2 and p-MAP3K7 co-expression in mice liver sections isolated from Flox, THKO- NTG, and THTG mice that were treated with a HFD for 16 weeks (magnification, ×40; n = 10 images per group for each staining) (**P < 0.01 vs. Flox HFD groups or NTG groups). b Representative immunoblotting bands for expression alterations of total amounts or phosphorylated forms of critical indicators associating with the Rhbdf2-MAP3K7 axis and its downstream events cascades including ADAM17, TNFR1/2, MKK7, p-MKK7, c-Jun, and p-c-Jun (Ser63) in the liver of 16-week HFD-fed Flox, THKO- NTG and THTG mice (n = 6 per experiment) (**P < 0.01 vs. Flox HFD groups or NTG groups). The GAPDH was used as a loading control. c Representative immunoblotting bands for expression changes of total amounts or phosphorylated forms of critical indicators associating with the Rhbdf2–MAP3K7 axis and its downstream events cascades including ADAM17, TNFR1/2, MKK7, p-MKK7, c-Jun, and p-c-Jun (Ser63) in primary hepatocytes isolated from the Flox, THKO- NTG, and THTG mice that were incubated with 400 μM PA for 10 h (n = 6 per experiment) (**P < 0.01 vs. Flox palmitate groups or NTG palmitate groups). The GAPDH was used as a loading control. d Representative immunoblotting bands for expression changes of total amounts or phosphorylated forms of critical indicators associating with the insulin signaling, including IRS1, p-IRS1(Ser307), AKT, p-AKT, GSK3β, p-GSK3β, FOXO1, and p-FOXO1, in the adenovirus-packed full-length Rhbdf2 sequences (AdRhbdf2) or shRNA targeting Rhbdf2 (AdshRhbdf2)-transfected THKO or THTG primary hepatocytes that were treated with 400 μM PA for 10 h (n = 4 per experiment) (**P < 0.01 vs. Palmitate/THKO-AdRhbdf2 or Palmitate/THTG-AdRhbdf2). The corresponding AdGFP was used as controls. The GAPDH was used as a loading control. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. Significance determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test (a–c) and Student’s two-tailed t test analysis (d).