Figure 1
Purification of ARIP4-containing complexes.
(A) FLAG-HA epitope-tagged ARIP4 (ARIP4-FH) was expressed and purified from HeLa cells by immunoprecipitation with an antibody specific to FLAG (lane 2), followed by an antibody specific to HA (lane 4). The purified proteins were resolved by NuPAGE gel electrophoresis and visualised by silver staining. As a control, a mock purification was performed on HeLa cells not expressing exogenous proteins (lanes 1 and 3). The ~54-kDa protein that directly associated with ARIP4-FH (indicated with an arrow), was identified as p62 by mass spectrometry analysis. Molecular weight standards are shown on the left. (B) FLAG-purified proteins were separated with a 10–40% glycerol gradient by centrifugation. The proteins in each fraction were purified with anti-HA antibody and the fractions (top to bottom) were resolved by NuPAGE gel electrophoresis and visualised by silver staining (upper panel) and immunoblotted with ARIP4-, p62- and Dyrk1A-specific antibodies (lower panels). (C) Interaction between endogenous p62 and ARIP4. HeLa nuclear extracts were immunoprecipitated with anti-ARIP4 antibody (A1 and A2 antibodies were produced by different rabbits) or rabbit IgG as a control. The immunoprecipitates were analysed by western blotting with the indicated antibodies.
