Figure 2

PARP1 regulated the protein stability of HIPK2. (a) The mRNA levels of both endogenous (endo, human) and exogenous (exo, mouse) HIPK2 were monitored by RT-PCR analysis following co-transfection of vectors for PARP1 (0.25 and 0.5 μg) or pLKO.1 PARP1 shRNA (shPARP1). (b and c) The protein levels of overexpressed and endogenous HIPK2 were examined following PARP1 knockdown in the presence of cycloheximide (20 μM). (d) HEK 293 cells were transfected with Myc-HIPK2 and empty vector or PARP1 expression vectors and then treated with 5 μM of MG132 for 10 h. The cells were then processed for immunoblotting as indicated. (e) Expression vectors for Myc-HIPK2 with or without HA-PARP1 were expressed in HEK 293 cells and then the transfected cells were treated with 5 μM of MG132. After 10 h, cells were further incubated with cycloheximide (20 μM) and collected at the indicated time points. The cells were analyzed by immunoblotting using anti-Myc and anti-HA antibodies. (f) The Myc bands in E were quantified by densitometry. Control (CTL) represents Myc-HIPK2 overexpression. Means±S.D. of three independent experiments are shown. (g and h) To examine the changes in the ubiquitination of HIPK2 after altering PARP1 expression, Myc-HIPK2 and HA-Ubiquitin (HA-Ub) expression vectors were transfected into HEK 293 cells with PARP1 expression vector or shPARP1. At 24 h after the transfection, the cells were treated with MG132 (5 μM) for 10 h. Next, the cell lysates were immunoprecipitated with anti-Myc antibodies, followed by immunoblotting using anti-Ub, anti-HA, anti-Myc and anti-PARP1. The input lane represents 10% of total cell lysates. Asterisks indicate the ubiquitinated Myc-HIPK2