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. 2004 Aug 24;2(9):e261. doi: 10.1371/journal.pbio.0020261

Figure 2. Rabankyrin-5 Stimulates Homotypic Fusion between Early Endosomes and Heterotypic Fusion between Early Endosomes and CCVs In Vitro.

Figure 2

(A) Rabankyrin-5 was efficiently immunodepleted from HeLa cytosol without affecting the EEA1 concentration. Twenty micrograms of HeLa cell cytosol, either treated with control IgGs and immunodepleted for Rabankyrin-5 or nontreated, was applied to SDS-PAGE and probed for the indicated antigens by Western blotting. Anti-γ tubulin was used as a loading control.

(B and C) Addition of recombinant Rabankyrin-5 stimulates homotypic and heterotypic fusion. Fusion (B) between early endosomes (EE-EE fusion) and (C) of early endosomes to CCVs (CCV-EE fusion) in vitro was performed in the presence of 3 mg/ml HeLa cytosol and an ATP-regenerating system (basal) and in the absence or presence of the indicated reagents. For some conditions, cytosol was immunodepleted for Rabankyrin-5 (columns 8–13) and in addition supplemented with anti–Rabankyrin-5 antibodies to neutralise the function of membrane-associated proteins (column 10). Background fusion activity is demonstrated by ATP (−Energy) or cytosol (−Cytosol) omission.