Fig. 7. UNC-53 binds SEM-5/GRB2. (A) Immunoprecipitation of in vitro produced UNC-53. Radiolabelled UNC-5371C protein, transcribed and translated in vitro, was immunoprecipitated with the MAb 16-48-2 under denaturing conditions, then incubated with protein G sepharose. The beads were washed and the bound products analysed by SDS-PAGE and fluorography (lane + Ab). As a control (lane – Ab), a reaction without MAb 16-48-2 was treated identically. The lower bands most likely correspond to proteins initiated at internal methionines, or arising from premature termination or proteolytic degradation. (B) GST pull-down assays. The radiolabelled UNC-5371C protein was incubated with SEM-5-GST (left panel) or GST (right panel) coupled to sepharose beads. After four washes, the remaining proteins bound to the beads were analysed by SDS-PAGE and fluorography. U, unbound; W1-4, washes 1 to 4; B, bound protein. (C) Western blot overlay assays. Cell lysates from bacteria containing the UNC-5371C-encoding plasmid pTB61, from induced (+) or uninduced (–) cultures were denatured in Laemmli buffer and the proteins separated by 5-25% gradient SDS-PAGE. Total proteins were revealed by Coomassie Blue staining (left panel). Additional gels were blotted to nylon membrane, incubated with biotinylated GST (middle panel) or biotinylated GST-GRB2 protein (right panel), and bound protein complexes subsequently detected using an alkaline phosphatase-linked anti-streptavidin-antibody and chromogenic substrate. The positions of the molecular weight markers (in kDa) are shown.