Figure 7

SmB' and SMN compete with coilin for Tim50a binding sites. Competition GST-pulldown experiments are shown. (A) SMN does not compete with SmB' for Tim50a binding sites. GST-Tim50aSac was incubated in separate reactions with SmB' (lane 2, top) or SMN (lane 2 bottom) followed by probing with anti-T7 (to detect SmB') and anti-SMN (to detect SMN). Lanes 3–6 show separate reactions in which a fixed amount of SMN was incubated with GST-Tim50aSac along with an increasing amount of SmB'. In lane 6, 27 times more SmB' was used in the reaction compared to the level of SMN. The input lanes account for 20% of the amounts used in all the reactions for SMN and the reactions shown in lane 2 and 3 for SmB'. (B) SmB' competes with coilin for Tim50a binding sites. GST-Tim50aSac was incubated with a fixed amount of the C-terminal coilin fragment (C214) and an increasing amount, in separate reactions, of SmB'. After Western blotting, anti-T7 was used to detect SmB' (top) and anti-coilin was used to detect C214 (bottom). Lane 4 shows the result of a reaction in which 3 times more SmB' compared to C214 was used in the binding assay with GST-Tim50aSac. Note that the level of recovered C214 is reduced in lane 4 compared to lane 2. The input lanes account for 20% of the amounts used in all the reactions for C214 and the reactions shown in lane 2 and 3 for SmB'. (C) Coilin competes with SMN for Tim50 binding sites. A competition experiment is shown wherein individual reactions were set-up containing a fixed amount of GST-Tim50aSac and SMN with increasing amounts of coilin fragment (C214). In lane 7, 27 times more coilin fragment was used in the reaction compared to the level of SMN. After SDS-PAGE and Western transfer, SMN and the coilin fragment were detected using antibodies specific to each protein. Lanes 2 and 3 show the binding of the coilin fragment or SMN to GST-Tim50aSac in the absence of competitor, respectively. The coilin fragment was added to the reactions containing GST-Tim5aSac and SMN starting at lane 4. As can been seen in lane 7, as more coilin fragment binds to GST-Tim50aSac, very little SMN is recovered. The input lane accounts for 20% of the amounts used in all the reactions for SMN and the reactions shown in lane 2 and 4 for the coilin fragment.