Figure 5.

TRIB3 interacts with ATF4 and negatively regulates ATF4. (A) Immunoprecipitation for TRIB3 in MRC5 cells. TRIB3 interacts with ATF4 by silver staining. (B) Co-IP of flag-tagged TRIB3 and HA-tagged ATF4 protein from the transfected cell lysates. (C,D) Overexpression of TRIB3 decreased the expression of ATF4 both at RNA and protein levels (N = 3 independent experiments). GAPDH was used as an internal control for qPCR, and β-actin was used as an internal control for western blot. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. ** p < 0.01. (E) Relative expression of ATF4 promoter-driven luciferase reporters in TRIB3-overexpressing cells (N = 3 independent experiments). Results were normalized to Renilla luciferase activity and expressed as relative luciferase units. Significant differences between groups were evaluated by two-way ANOVA with Šídák’s multiple comparisons test for pairwise comparisons. *** p < 0.001. ns, no significance.