Tbata interaction with Uba3 blocks formation of the Nedd8 E1 and inhibits cell division. (A) GST pulldown assays were done with GST, GST-Ttv3 (T3), and GST-Ttv5 (T5) beads added to equal amounts of radiolabeled Myc-Uba3 and human AppBp1. Anti-Myc immunoprecipitations were used as a positive control using either Myc-Uba3 mixed with AppBp1 alone (IP) or from the supernatants of GST-Ttv3 (IP T3 sup) or GST (IP GST sup) pulldown assays (bottom). The asterisk shows the position of a protein differing by 8.7 kD produced during in vitro translation of Uba3 cDNA caused by usage of different ATG codons (Gong and Yeh, 1999). (B) The mif-inducible cell lines, EGFP-C and Ttv3-C were treated with mif (10−8 M) to express EGFP (EGFP-C+mif) and Ttv3-EGFP fusion protein (Ttv3-C+mif), respectively. The whole-cell lysates were immunoprecipitated and immunoblotted as indicated. The blot was stripped and reprobed with rabbit nedd8 antibody (middle). (C) HEK 293 cells were transfected with 0.25 mg Ttv3-EGFP and different amounts of either Uba3 or D-uba3 DNA (◇, 0 mg; ▪, 1.25 mg Uba3; △, 1.25 mg D-uba3; ▲, 2.5 mg Uba3; ○, 2.5 mg D-uba3). Control (◆) was transfected with 0.25 mg EGFP. Absolute cell numbers were measured at indicated times after transfection. Data shown is representative of five independent experiments done in triplicate. (D) HEK 293 cells were transfected with indicated plasmids alone on in a combination at 1:10 ratio. T3, Ttv3-EGFP; Uba, Uba3; D-Uba, D-Uba3.
