Figure 2

SNIPER(TACC3) induces ubiquitylation and proteasomal degradation of TACC3. (a) HT1080 cells were treated with 30 μM of SNIPER(TACC3)-1 or -2 in the presence or absence of 25 μM of MG132 for 4 h. Whole-cell lysates were analyzed using western blotting with the indicated antibodies. The numbers below the TACC3 panels represent the TACC3 level relative to the respective control normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (b) HT1080 cells were co-transfected with the expression vector of Flag-TACC3 and/or HA-ubiquitin. After 40 h, cells were treated with the indicated compounds in the presence of 25 μM of MG132 for 3 h. Cells were lysed, and Flag-TACC3 was immunoprecipitated with an anti-FLAG antibody. The ubiquitylated TACC3 was detected with an anti-HA antibody (left panels) or an antibody specific to K48-linked ubiquitin (right panels). DMSO, dimethyl sulfoxide; IB, immunoblot; IP, immunoprecipitation