Figure 6.

STUB1 promoted ubiquitination and degradation of RUNX1–RUNX1T1. A, 293T cells were transfected with Myc–ubiquitin, HA–RUNX1–RUNX1T1, and/or FLAG–STUB1. Nuclear and cytoplasmic fractions were isolated and immunoprecipitated with anti-HA antibody, following the detection of ubiquitinated RUNX1–RUNX1T1 with anti-Myc antibody. STUB1 induced ubiquitination of RUNX1–RUNX1T1 mainly in the nucleus. s.e., short exposure; l.e., long exposure. B, 293T cells were transfected with HA–RUNX1–RUNX1T1 and/or FLAG–STUB1. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody, following the detection of STUB1-bound RUNX1–RUNX1T1 with anti-HA antibody. C, Kasumi-1 cells were retrovirally transduced with a vector control or FLAG–STUB1 (coexpressing GFP). GFP⁺ cells (transduced cells) and GFP⁻ cells (nontransduced cells) were sorted, and cell extracts were analyzed with anti-RUNX1, anti-FLAG, and anti-Tubulin antibodies. Enforced expression of STUB1 in Kasumi-1 cells resulted in down-regulation of RUNX1 and RUNX1–RUNX1T1 expression. IB, immunoblot.