Figure 8

Tim50a interacts with SMN and snRNPs in vivo. (A) SMN and Tim50a form a complex in vivo. HeLa cells were transfected with empty GFP vector or GFP-tagged Tim50a and Tim50aN87. Lysate was subjected to immunoprecipitation with polyclonal anti-GFP antibodies, followed by Western blotting and probing with anti-SMN antibodies (top panel). The blot was reprobed with monoclonal anti-GFP (lower panel) to visualize the level of immunoprecipitated GFP, GFP-Tim50a and GFP-Tim50aN87. (B) Tim50a and Tim50 form a complex with snRNPs. HeLa cells were transfected with empty GFP vector, GFP-tagged Tim50a or GFP-Tim50 and lysate was subjected to immunoprecipitation with anti-Sm (Y12) antibodies, followed by Western blotting and probing with anti-GFP antibodies. Immunoglobulin heavy IgG(H) chains are marked. The input lanes represent 2% of the amount of lysate used in the immunoprecipitation reactions and have been overexposed five-fold relative to the immunoprecipitation reactions in order to visualize the GFP-Tim50a signal. (C) Tim50a and Tim50 bind the Sm protein, SmB'. HeLa cells were transfected with myc-tagged Tim50a or Tim50 and lysate was incubated with beads loaded with GST or GST-SmB', followed by Western blotting and probing with anti-myc antibodies. The input lanes represent 2% of the amount of lysate used in the pulldown reactions. Approximately equal amounts of GST and GST-SmB' proteins were used.