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Fig. 1. SCAMPs interact with NHE7. (A) Schematic representation of SCAMP1, 2 and 5 showing the Asn-Pro-Phe (NPF) repeat and a highly conserved region (underlined) containing a Pro-rich (P rich) motif and four transmembrane (TM) segments. (B) CHO cells were transiently transfected with HA-tagged NHE7 or NHE6 and myc-tagged SCAMP1, 2 or 5. Cells were lysed in 0.5% NP40/PBS for 30 minutes on ice and lysates were cleared for 20 minutes at 16,000 g at 4°C. Cell lysates (Lys) were then immunoprecipitated with mouse anti-HA antibody (HA) or pre-immune serum (con) and bound SCAMPs were detected on western blots probed with rabbit anti-myc antibody. 5% of the total lysate was loaded. (C) GST and GST fused with NHE7 C-terminal tail (GST-NHE7 [542-725]) were expressed in E. coli and purified by incubation with glutathione-conjugated sepharose beads. 2 µg immobilized GST or GST-NHE7 C-terminal fusion protein was incubated with PC12 cell lysate, and any SCAMPs bound to the GST fusion protein were detected by western blotting.
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