Fig. 1. Incorporation of GFP-tagged MVP molecules in vaults. (A) The SW1573 transfectant expressing MVP-GFP (a) was examined by fluorescence microscopy. The cell line SW1573 (b) was processed for indirect immunofluorescence and stained with anti-MVP (LRP-56). Bar, 10 µm. (B) Vault particles were pelleted from a total cell lysate (TL) of SW1573, SW1573/MVP-GFP transfectants and SW1573 transfectants expressing a GFP-tagged truncated MVP (SW1573/MVP706-GFP) by a 100,000 g centrifugation step. Immunoblotting using rabbit polyclonal anti-MVP was performed to determine the presence of MVP and the GFP-fusion proteins in the resulting pellet (P) and supernatant (S) fractions. Note that SW1573/MVP-GFP and SW1573/MVP706-GFP show two bands, representing endogenous (lower band) and GFP-tagged MVP or truncated MVP (upper band). (C) Immunoblot analysis showing the presence of the minor vault protein VPARP in immunoprecipitates obtained from MVP-GFP transfectants using anti-GFP (lane 2). Lane 1 contains control immunoprecipitates in which a polyclonal rabbit pre-immune serum was used.

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