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Figure 1 | Molecular Neurodegeneration

Figure 1

From: FE65 as a link between VLDLR and APP to regulate their trafficking and processing

Figure 1

FE65 interacts with VLDLR in COS7 cells and brain lysates. A. COS7 cells were transiently transfected with VLDLR (HA-tagged on the C terminal) and FE65 (myc-tagged on the C terminal), or each construct alone with empty vector. Cell lysates (200 ug) were immunoprecipitated with anti-myc and the precipitate was probed with anti-HA. FE65 co-precipitated with VLDLR in COS7 cells (lane 3, upper panel). Cell lysates (20 ug) were probed for VLDLR and FE65 to demonstrate consistent levels of expression across each condition (middle and lower panel). B. COS7 cells were transfected with VLDLR (HA-tagged) and FE65 (myc-tagged), or each construct alone. Cell lysates (200 ug) were immunoprecipitated with anti-HA and the precipitate was probed with anti-myc. VLDLR co-precipitated with FE65 in COS7 cells (lane 3, upper panel). Cell lysates (20 ug) were probed for VLDLR and FE65 to demonstrate consistent levels of expression across conditions (middle and lower panel). C. VLDLR deletion construct expressing the CTF of VLDLR with an HA tag was generated. D. COS7 cells were transiently transfected with VLDLR or VLDLR CTF constructs. Cell lysates (20 ug) were probed for anti-HA to verify the expression of constructs. E-F. COS7 cells were transiently transfected with full length VLDLR and empty vector, full length VLDLR and FE65 or VLDLR CTF and FE65. E. Cell lysates (200 ug) were immunoprecipitated with anti-myc and the precipitate was probed with anti-HA. VLDLR CTF and full length VLDLR co-precipitated with FE65 in COS7 cells (lane 2 and 3, upper panel). Cell lysates (20 ug) were probed for FE65 to demonstrate consistent levels of expression across each condition (lower panel). F. Cell lysates (200 ug) were immunoprecipitated with anti-HA and the precipitate was probed with anti-myc. FE65 immunoprecipitated with both VLDLR constructs consistent with the experiment in E. G. Mouse brain lysates (100 ug) were immunoprecipitated with 5F3 (VLDLR antibody) or Ig G and probed with anti-VLDLR (upper panel) and anti-FE65 (lower panel). H. Mouse brain lysates (100 ug) were immunoprecipitated with anti-FE65 or IgG and probed with anti-FE65 (upper panel) and anti-VLDLR (lower panel). I. Schematic of recombinant GST-VLDLR CTF J. The recombinant GST-VLDLR CTF was expressed in E.coli strain BL21, using the pGEX system as indicated. The GST fusion proteins were then purified using glutathione-agarose beads (Sigma), in accordance with the manufacturer's instructions. (I, total cell extract of induced cells; S, supernatant of sonicated extracts of induced cells (soluble recombinant protein); P, Pellet of sonicated extracts of induced cells (insoluble recombinant protein); E, Purified VLDLR CTF proteins). K. Recombinant GST-VLDLR CTF proteins were immobilized onto GSH-agarose. Wild-type brain lysates were then subjected to GST-pull-down experiments with either immbolized GST (lane 1, 3) or immobilized GST-VLDLR CTF (lane 2, 4) and probed for FE65.

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