Figure 1
SAP130 is a CSN1 interacting protein. (A) Diagram of bait HT-FS1-NTD. From the N-terminus, this protein contains a His-tag (6 × HIS), Tobacco Etch Virus protease recognition and cleavage site (TEV site) and Flag tagged CSN1-NTD (1–196 aa). (B) Coomassie Blue staining gel showing the input NIH3T3 cell lysate and proteins eluted after TEV protease cleavage. Identity of corresponding proteins is labeled. (C) SAP130-myc was co-transfected in HeLa cells with empty vector (lane 1) or Flag-tagged CSN subunits as indicated. Total cell extract and the Flag immunoprecipitated (IP) proteins were probed with anti-myc antibody. (D) CSN1 was immunoprecipitated from DRB treated NIH3T3 nuclear extract. Endogenous SAP130 was enriched in CSN1 IP compared to pre-immune serum (pre-im). (E) HA-SAP130 was in vitro translated and S35-Met labeled in rabbit reticulocyte lysate. The samples were incubated with recombinant GST or GST-CSN1 proteins (1 μg each) for 30 min. Following GST pull-down, samples were analyzed by autoradiogram (upper panel) or anti-GST immunoblotting (lower panel). (F) HA-CPSF160 was transiently co-transfected in HeLa cells with empty vector (lane 1) or Flag-tagged CSN subunits. Total cell extract and the Flag immunoprecipitated proteins were probed with anti-HA antibody.