Figure 5.

DeSI-1 modulates the transcriptional repressor activity of BZEL. (A) Raji B cells were transfected with reporter genes driven by the blimp-1 promoter along with a combination of expression plasmids encoding BZEL (7 μg), DeSI-1 (7 μg) and DeSI-1^C108S (7 μg). As a control for the transfection efficiency, the cells were also cotransfected with pCMV-β-galactosidase control vector. Subsequently, cell lysates were assayed for luciferase activity, and the luciferase activity was normalized against β-galactosidase activity. Values are the averages of three independent experiments performed in duplicate. Error bars represent standard deviations. (B) A20 B cells were electroporated with reporter genes driven by the blimp-1 promoter along with a combination of expression plasmids encoding BZEL (2 μg), shDeSI-1–1 (10 μg), shDeSI-1–2 (10 μg) or control shRNA (10 μg). Subsequently, luciferase activity was determined as described in A. Values are the averages of three independent experiments performed in duplicate, and error bars representing standard deviations are shown. Data were also analysed by Student's t-test; *P<0.005 and **P<0.02. (C) Cell lysates from the experiment shown in B were subjected to immunoprecipitation (IP) with anti-BZEL antibody and subsequently to immunoblot (IB) analysis with anti-SUMO1 (upper panel) or anti-BZEL (lower panel) antibodies (left). As a control for DeSI-1 expression, whole-cell extracts (WCE) were analysed by immunoblotting with anti-DeSI-1 or anti-actin antibody (right). BZEL, BTB-ZF protein expressed in effector lymphocytes; DeSI-1, DeSumoylating Isopeptidase 1; shRNA, short hairpin RNA; SUMO1, small ubiquitin-like modifier 1.