Fig. 5

TRIM69 negatively regulates the expression of c-Jun.

(A) HEK 293T cells were transfected with Flag-TRIM69 or a control expression vector. The cell lysates were analyzed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. (B) HEK 293T cells were transfected with Flag-TRIM69 or a control expression vector. At 36 h post-transfection, the cells were treated with cycloheximide (200 µg/ml, CHX) and MG132 (20 µM) or chloroquine (100 µM) for 2 h. (C) HEK 293T cells were co-transfected with Flag-TRIM69 along with siRNA-NC or siRNA-TRIM69. The cell lysates were analyzed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. (D,E) SH-SY5Y and HEK 293T cells were transfected with siRNA-NC or siRNA-TRIM69. The cell lysates were analyzed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. Simultaneously, the relative RNA level of TRIM69 was determined by qPCR. Data were measured in triplicate and statistically analyzed by unpaired t-test, p < 0.01; values and bars represent the mean and standard deviation, respectively; (F,G) trim69 knockdown decreased the expression of c-Jun in zebrafish. trim69-CON: control group; trim69-MO: trim69 knocking down; scale bar: 200 µm.