Figure 2.
PrPc immunoprecipitates specifically with human soluble Aβ dimers. A, Potential interaction of PrPc with Fyn in AD brain tissues was assessed by SDS-PAGE analysis of the PrPc/caveolin-1/Fyn complex following immunoprecipitation of PrPc (with 8B4 or C20 antibodies) or Fyn. The gray arrowheads indicate exogenous antibodies used for IP. B, PrPc forms a putative complex with Fyn and soluble Aβ dimers using AD brain tissue. Western blots for Aβ were performed with 6E10. Following membrane stripping, PrPc and Fyn were revealed with PrP C20 and Fyn3 antibodies. Additional IPs with different cases are shown on the right panels to illustrate the reproducibility of the findings. C, Western blot analysis of TBS-soluble extracts from selected brains of subjects with AD or no cognitive impairment (N). Monomers, dimers, and trimers are readily detected using 6E10. D, Immunoprecipitation of PrPc with exogenous human Aβ dimers in a cell-free assay. Affinity-purified human soluble Aβx-40/42 species (lane 1, left and right panels, an estimated total of 2.85 ng relative to Aβ1–42 standards) were added to brain protein extracts with no detectable Aβ (N; same as A); PrPc was immunoprecipitated using the C20 antibody and bound Aβ species were detected with 40/42-end specific antibodies (top left panel) or 6E10 (bottom left and right panels). Full Western blot images indicated that isolated large soluble Aβ assemblies (e.g., Aβ*56 and protofibrils) were not pulled down by C20 in our assay (right panel). IP, immunoprecipiration; No Ab, no antibody; N, nonimpaired age-matched control brain; AD, Alzheimer's disease brain; oAβ, endogenous oligomeric Aβ species purified from human AD brain tissue; sAβ1–42, 0.5 ng of human synthetic Aβ1–42 (Sigma-Aldrich).