Figure 4. USP28 deubiquitinates LSD1.

(A) Myc-LSD1 was co-expressed with vector or Flag-USP28 in HEK293 cells. After treating cells with cycloheximide (CHX) for indicated time intervals, expression of LSD1 and USP28 was analyzed by Western blotting (top panel) using Myc and Flag antibodies, respectively. The intensity of LSD1 expression for each time point was quantified by densitometry and plotted (bottom panel). Experiment was repeated three times and a representative experiment is presented. Exp stands for exposure.

(B) HT29 cells were transfected with control or USP28 siRNA. After cells were treated with CHX, expression of endogenous LSD1 and USP28 was analyzed by Western blotting (top panel); the intensity of LSD1 expression for each time point was quantified by densitometry and plotted (bottom panel). Experiment was repeated three times and a representative experiment is presented.

(C) Myc-LSD1 and HA-ubiquitin were co-expressed with wild-type or catalytic inactive (CI, C171A) mutant of USP28 in HEK293 cells. After cells were treated with or without 10 μM MG132 for 6 h, LSD1 was immunoprecipitated and the poly-ubiquitination of LSD1 was detected by Western blotting using HA antibody. Immunoprecipitated LSD1 was blotted using Myc antibody.

(D) BT549 and MCF7 cells stably transfected with control, USP28 shRNA were treated with or without MG132 for 6 h. Extracts were immunoprecipitated with LSD1 antibody and the poly-ubiquitination of LSD1 was examined by Western blotting using ubiquitin antibody.

(E) Ubiquitinated LSD1 was purified from MG132 treated HEK293 cells expressing Myc-LSD1, and then incubated with purified Flag-tagged wild-type USP28 or CI-USP28 in a deubiquitination assay as described in Experimental Procedures. The poly-ubiquitinated state of LSD1 was examined by Western blotting using HA antibody. Immuno-purified LSD1 and USP28 used in this assay were analyzed using Myc and Flag antibodies, respectively.