Figure 7.

Association of PRP31, NUFIP and ZNHIT3 with the SMN complex. (A) Western blotting of inputs and pellets of anti-GFP immuno-precipitates of 293T cells transfected with the indicated GFP constructs. Left gel: no RNase treatment; right gel: RNase treated samples. Membranes are probed with the indicated antibodies. (B) Autoradiogram of a gel with input and pellets of immune-precipitates of in vitro translated [³⁵S]methionine-labeled Ran, SMN and NUFIP (Total; 10%), tested for binding to immobilized SMN complex (SMN complex; 100%). Specific binding was tested with anti-Flag beads pre-incubated with HeLa cell extracts expressing Flag-Gemin2. Non-specific binding was assessed with anti-Flag beads incubated with parental cell extracts that do not express Flag-Gemin2 (Control). (C) Micrographs of yeast strains grown in drops on yeast-two hybrid selective media. pACTII-NUFIP was used as prey and the components of the SMN complex used as bait (left panel). Controls were performed with empty pACT2 vector (right panel). Growth on medium lacking histidine indicates an interaction. Increasing amount of 3-Amino-1,2,4-triazol (3-AT), was added to the medium to evaluate the strength of the interaction. (D) Western blots of inputs (Total; 10%) and immuno-precipitates (100%) of E. coli total extracts expressing recombinant Gemin3, 4, 6 or 15.5K as control, and blotted with the indicated antibodies. Immuno-precipitation was performed in presence of RNase and with beads containing immobilized recombinant GST-NUFIP or GST as control.