Interaction of LRP1 and NYGGF4 expressed in mammalian cells. A. COS-7 cells were transfected with vector (pcDNA3) or NYGGF4-V5 cloned into pcDNA3.1V5/His and resultant cell lysates were subjected to GST-pulldown with either GST or with GST-LRP1 (cytoplasmic domain only), as indicated by the scheme at the base of the figure. Aliquots of cell lysates (right most lanes) and aliquots of the GST-pulldowns were probed by immunoblotting with anti-V5 antibody for NYGGF4. NYGGF4 is precipitated with GST-LRP1, but not with GST alone. B. FLAG-tagged mLRP4 and V5-tagged NYGGF4 were cotransfected into H4 neuroglioma cells, and immunoprecipitation (IP) of either protein by FLAG or V5 antibody successfully coprecipitated the other protein as detected by immunoblot (IB). Lysate was also blotted to show expression in transfected cells (Input) and control immunoprecipitations were carried out with purified control immunoglobulin (IgG). C. FLAG-tagged mLRP4 and V5-tagged NYGGF4 were cotransfected into either H4 (upper) or Neuro 2A (lower) cells, and detected by immunocytochemistry. Both proteins showed extensive colocalization, especially in the perinuclear region. Results are representative of three independent experiments.