Fig. 3.

Interaction between Tau and fragment F3*. (a and b) Aggregation of full-length Tau nucleated by tau fragment F3*. (a) Blot analysis (antibody K9JA) of aggregation of full-length Tau* (htau40ΔK280) induced by F3*. Without the fragment, Tau* remains in supernatant (lanes 1 and 2), but the fragment induces pelletable aggregates comprising both components (lanes 3 and 4). The same result, but with lesser efficiency, is obtained with WT full-length Tau, i.e., no aggregation for Tau alone (not shown) but aggregate formation in the presence of F3* (lanes 5 and 6). (b) Aggregation of full-length mutant tau (Tau*) in inducible N2a cells demonstrated by ThS staining. (Left) tau expression monitored by immunolabeling with antibody K9JA (Top and Middle) or SA4473 (epitope in the first N-terminal insert of tau) and Cy5 secondary antibody (Bottom). (Center) Formation of aggregates monitored by ThS staining; note that it occurs only in the presence of F3* fragments. (Right) Merged images. The staining with antibody SA4473 confirms that full-length tau is present in aggregates. For analogous results on P301L mutants see SI Fig. 6. (c and d) Interaction of F3* with Tau* in N2a cells. (c) Separation of sarkosyl pellet from N2a cells by iodixanol gradient centrifugation, demonstrating coaggregation of full-length Tau* with F3*. Note that Tau* appears only in fractions enriched in F3* and that higher aggregates are present in the stacking gel in fractions 4–7. (d) Interaction of F3* with Tau* by coimmunoprecipitation. F3* expressed alone or coexpressed with Tau* in N2a cells was pulled down with antibody SA4473 and analyzed by SDS/PAGE followed by immunoblotting with K9JA antibody. Note that part of F3* was pulled down by SA4473 when it was coexpressed with Tau*.