Figure 4: Site-specific Ets-1 deubiquitination by Usp9x.

(a) HEK293T cells ectopically expressing HA-Ets-1 (WT), HA-Ets-1/K388A or HA-Ets-1/K388R co-expressed with HA-Ub were subjected to immunoprecipitation with Ets-1 antibody (Bethyl) followed by immunoblotting for HA (top) or Ubiquitin (bottom). Ets-1 in the pulldown was also immunoblotted with anti-Ets-1 (Bethyl—bottom). (b) HEK293T cells ectopically expressing HA-Ets-1 alone or co-expressed with FLAG-Usp9x and HA-Ub (as noted) were subjected to HA (Ets-1) immunoprecipitation followed by immunoblotting of Ubiquitin (top). Whole cell lysates (WCL) were also immunoblotted for the protein indicated (bottom). (c) BRAF mutant SK-Mel29 cells were stably transfected with HA-Ets-1 WT or the K388R mutant plasmid, treated with 30 μg ml⁻¹ of cycloheximide (CHX), and harvested at the time points indicated after CHX addition. Immunoblot for HA (Ets-1) is shown. (d) The blot from c was subjected to densitometric scanning (ImageJ software) to detect changes in HA-Ets-1 protein levels over time. (e) Immunoblot for HA and actin in SK-Mel29 cells stably expressing HA-Ets-1 WT or HA-Ets-1/K388R. Protein expression levels were quantified by densitometry (ImageJ software). (f) Colony growth (detected by crystal violet staining) of SK-Mel29 cells expressing control, HA-Ets-1 WT or HA-Ets-1/K388R and grown 21 days in standard 2D culture. (g) Phase contrast images of SK-Mel29 cells expressing control, HA-Ets-1 WT or HA-Ets-1/K388R and grown on matrigel for 7 days. (h) Quantification of growth of colonies in (f) after 21 days. All data shown are mean values±s.d. (error bar) from three replicates.