Fig. 3. Ubiquitylation of RUNX1. P19 cells were transfected with the plasmids of expression vector alone or plasmid expressing RUNX1 tagged with HA at the N-terminus and His₆ at the C-terminus. Twenty-four hours after transfection, the cells were treated with lactacystin and incubated for an additional 12 h. RUNX1 proteins were purified from the cell extract by NTA resin under denaturing conditions. Western blotting was performed with anti-HA (A) or anti-ubiquitin (ub) (B): lane 1, vector alone without lactacystin; lane 2, vector alone with lactacystin; lane 3, HA-tagged RUNX1 without lactacystin; lane 4, HA-tagged RUNX1 with lactacystin. The positions of RUNX1 and ubiquitylated RUNX1 [(Ub)n-RUNX1] are indicated by arrows.