Fig. 4.

JP3 inhibits angiogenesis through degrading SP1 by E3 ubiquitin ligase TRIM25 in GC cells. a The relationship between the ubiquitin enzymes and SP1 was predicted online (http://genemania.org/). b BGC823 cells were treated with JP3 (0, 1, 10, 50 μM) for 24 h. The indicated protein levels were determined by Western blotting. c-d BGC823 (c) and SGC7901 (d) cells were pre-treated with MG132 (10 μM) for 6 h, and the endogenous protein-protein interaction between TRIM25 and SP1 was assessed by IP with anti-TRIM25 or anti-SP1 antibodies, followed by Western blotting. e si-TRIM25 was transfected into BGC823 cells for 48 h, followed by JP3 treatment for 24 h, and then, tube formation assay was performed. f The tube number was analyzed (means ± SEM, n = 3). ***P < 0.001. g BGC823 cells were co-transfected with His-Ub and si-TRIM25 or si-con for 48 h, and then treated with or without JP3 (50 μM) for another 24 h, followed by pre-treatment with MG132 (10 μM) for 12 h. Ubiquitinated SP1 was determined by IP with anti-SP1. The indicated protein levels were determined by Western blotting. h-i BGC823 cells were co-transfected with His-Ub and HA-TRIM25 or HA-con for 48 h, followed by pre-treatment with MG132 (10 μM) for 12 h, and then, tube formation assay was performed. Ubiquitinated SP1 was determined by IP with anti-SP1