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Fig. 6. NHE7 and SCAMPs co-fractionate by sucrose equilibrium density centrifugation. Homogenate was prepared from PC12 cells stably transfected with 1D4-tagged NHE7 and analyzed by sucrose equilibrium density centrifugation. Fifteen fractions were taken from the top and an equal volume of each fraction was analyzed on a western blot probed with anti-1D4 and different antibodies that recognize endogenous proteins. The following organellar markers were used: GM130 (cis/medial-Golgi), syntaxin 6 (Stx6; TGN/TGN-derived vesicles), syntaxin 13 (Stx13; recycling endosome), Rab 11 (recycling endosome), transferrin receptor (TfR; recycling endosome), ß-COP (cis-Golgi), synaptophysin (Syp; synaptic-like microvesicles), PDI (ER) and EEA1 (early endosomes). Note that NHE7 and SCAMPs showed similar peaks in fractions 7, 8 and 9, but SCAMPs have broader distribution (fractions 5 and 6). In SCAMP2, a less prominent 35 kDa band (labelled with asterisks) was observed in addition to the expected 38 kDa band. This minor band probably represents a non-specific reaction, but could also reflect a proteolytically cleaved form.





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