Figure 2
SAP130 interacts with Nedd8 modified cullins. (A) Whole cell extracts (lysate) and anti-HA immunoprecipitate (IP HA) from HEK293 cells transiently transfected with empty vector (-) or HA-SAP130 (+) were analyzed by immunoblotting using the antibodies against cullins or SAP155, a component of SF3b RNA-splicing complex. (B) Rabbit reticulocyte lysate (RRL) containing in vitro translated HA-SAP130 were incubated with recombinant GST-Nedd8, GST-Ub or GST (1 μg each). HA IP samples were analyzed by anti-Cul2 and anti-GST blots and autoradiogram (S35). (C) HA-SAP130 was in vitro translated in RRL supplemented with GST or GST-Nedd8 (1 μg each). Anti-GST blot shows that free GST-Nedd8, like GST alone, was not associated with HA-SAP130. (D) HeLa Cells were transfected with siRNA reagent against CAND1 or laminin-A control. Input lysate or HA IP samples were blotted with anti-CAND1 or Cul2 antibodies. Note that there was weak non-specific binding of un-neddylated Cul2 to HA beads (lane 1).