Fig. 2. FEH-1 and APL-1 maintain the binding properties of mammalian homolog proteins. (A) C. elegans (lanes 1-3) or mouse brain (lane 4) lysates were resolved by SDS-PAGE and probed with pre-immune serum (lane 1), pre-adsorbed (lane 2) or purified FEH-1 antibody (lane 3) or anti-Fe65 serum (lane 4). In lanes 5 and 6, immunoprecipitated FEH-1 treated (lane 6) with calf intestine alkaline phosphatase (CIP) or untreated (lane 5) was assayed by western blot analysis with FEH-1 antibody. (B) Glutathione-sepharose beads were saturated with wild-type GST (lane 1) or with fusions of GST with the APL-1 cytodomain (GST-APL-1, lane 2), the FEH-1 PTB1 (GST-PTB1, lane 3) or PTB2 (GST-PTB2, lane 4) domains and incubated with 500 µg each of C. elegans proteins. Bound proteins were resolved by SDS-PAGE and probed with FEH-1 antibodies. Lanes 1' to 4' show Ponceau S staining of the filter to indicate the amounts of each GST proteins used. (C) Glutathione-sepharose beads were saturated with wild-type GST (lane 2) or with fusions between GST and the FEH-1 PTB2 domain (GST-PTB2, lane 3); the FEH-1 PTB1 domain (GST-PTB1, lane 4) and were incubated with 2 mg of C. elegans proteins. Bound proteins were resolved by SDS-PAGE and probed with APL-1 antibodies. Lane 1 contains a C. elegans protein lysate sample. Lanes 2'-4' show Ponceau S staining of the filter.