Fig. 5: TRIM22 binds to Raf-1 and regulates SPHK2/MAPK signaling through its RING domain.
a Luciferase activity in U251MG and A172 cells transfected with different TRIM22 truncation mutants (n = 3 per group). U251MG: Finteraction(5, 24) = 145.2, P < 0.0001; A172: Finteraction(5, 24) = 113.2, P < 0.0001. b The effects of different TRIM22 truncation mutants on the core protein of the SPHK2/MAPK pathway were detected by Western blotting in U251MG and A172 cells. c Western blot analysis and quantification of Raf-1 protein in different modified cells treated with cycloheximide (CHX; 25 μg/mL) for 0, 8, 16, and 24 h (n = 3 per group). U251MG: Finteraction(6, 24) = 30.31, P < 0.0001; A172: Finteraction(6, 24) = 75.78, P < 0.0001. d Ubiquitination of Raf-1 in an in vitro assay. e Ubiquitination assay of Raf-1 in modified U251MG, A172 and TJ905 cells. f Co-IP was used to detect the exogenous binding of TRIM22 and Raf-1 in 293 T cells transfected with Flag-TRIM22 and HA-Raf-1. g Exogenous binding of TRIM22 and Raf-1 in U251MG and A172 cells using anti-TRIM22 and anti-Raf-1 antibodies. h Schematic representation of wild-type TRIM22 and Raf-1 and the indicated deletion mutants. Western blot analysis of Co-IPs in 293 T cells transfected with Flag-TRIM22/HA-Raf-1 alone or together with the indicated HA-Raf-1/Flag-TRIM22 constructs. The data were analyzed using two-way ANOVA, and all data are expressed as the mean ± standard deviation. *P < 0.05 represents a statistically significant difference between the two groups. ns, not significant. Each experiment was repeated three times. EV: empty vector.
