T310A mutation decreases TSP50’s ability to interact with NF-κB:IκBα complex and to promote NF-κB signaling.

<p>(A) Interaction between TSP50 and TSP50 T310A with NF-κB:IκBα complex. Equal amounts of protein from the cytoplasmic fraction of cells and immunoprecipitates with anti-TSP50 antibodies were subjected to immunoblotting with antibodies as indicated. (B) and (C) Effects of TSP50 or TSP50 T310A on NF-κB activation. pcDNA3 cells, pcDNA3-TSP50 cells and pcDNA3-TSP50 T310A cells were treated with 100 ng/ml of PMA for 15 min, and then (B) IκBα protein levels in these cells or (C) p65 protein levels in the nuclear fractions were detected by Western blotting, and quantitated using ImageJ software. GAPDH was used as a loading control. Experiments were carried out in triplicate. (D) NF-κB luciferase reporter assay. HEK293T cells were transiently transfected with pNF-κB-luc plasmid along with pcDNA3-TSP50, pcDNA3-TSP50 T310A, or pcDNA3 empty vector, respectively. Forty-eight hours after transfection, cells were pretreated with 400 ng/ml of PMA for 15 min and luciferase activity was assayed. The results were expressed as the fold activity in the luciferase activity of each sample versus that of the vector control. Three independent replicates of each experiment were performed. (E) NF-κB CHIP assay. L02 cells transfected with pcDNA3 empty vector, pcDNA3-TSP50, and pcDNA3-TSP50 T310A were treated with 400 ng/ml of PMA for 15 min before harvesting. The anti-p65 antibodies were used for immunoprecipitation and cyclin D1 promoter fragments were amplified and quantitated using ImageJ software. (F) Effects of TSP50 and TSP50 T310A on the PMA-induced expression of NF-κB target gene. L02 cells transfected with pcDNA3 empty vector, pcDNA3-TSP50 or pcDNA3-TSP50 T310A were treated with 100 ng/ml of PMA for 4 h, and then cytoplasmic extracts were analyzed by Western blotting using antibodies against cyclinD1 and GAPDH. (G) and (H) Effects of TSP50 or TSP50 T310A on the PMA-induced expression of NF-κB target genes. pcDNA3 cells, pcDNA3-TSP50 cells, and pcDNA3-TSP50 T310A cells were treated with PMA (100 ng/ml) for 4 h, and then cytoplasmic extracts were analyzed by Western blotting using antibodies against (G) COX-2, (H) C-myc, and GAPDH.