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. 1999 Aug 1;19(15):6538–6548. doi: 10.1523/JNEUROSCI.19-15-06538.1999

Fig. 5.

Fig. 5.

Recombinant overexpression of caveolin-3 stimulates the β-secretase processing of APP. A–D, The medium and/or cell lysates from COS-7 cells cotransfected with APP and caveolin-3 were subjected to immunoprecipitation and immunoblot analysis or ELISA. Mock-transfected cells and/or cells transfected with APP alone were processed in parallel. A,Top, The total amount of sAPP in the medium was measured by immunoprecipitation and immunoblotting using an anti-HA antibody. Note that sAPP was significantly increased in cells cotransfected with APP and caveolin-3. Middle, Cell lysates were probed with anti-HA antibodies to assess the total amount of APP expressed. Note that the amount of APP expression is similar in cells transfected with APP alone or in cells cotransfected with APP and caveolin-3 (compare lanes 2, 3).Bottom, Cell lysates were also probed with anti-myc to detect expression of caveolin-3. B, Top, The amount of sAPPα in the medium was measured by immunoprecipitation with the anti-Aβ antibody and subsequent immunoblot analysis with the anti-HA antibody. Note that sAPPα was detected only in medium harvested from cells that were transfected with APP alone but not in medium from cells cotransfected with APP and caveolin-3. Bottom, Cell lysates were probed with anti-HA antibodies to detect intact APP. Note that the amount of intact APP is similar in cells transfected with APP alone or in cells cotransfected with APP and caveolin-3 (compare lanes 1,2). C, Top, The C-terminal fragments of APP generated by α-secretase cleavage (αAPPct) or β-secretase cleavage (βAPPct) were detected by immunoprecipitation with the antibody 4G8 and immunoblot analysis with antibodies directed against the C-terminal FLAG epitope attached to APP. Note that in cells cotransfected with APP and caveolin-3 that the levels of bothβAPPct and αAPPct were increased.Bottom, Cell lysates were immunoprecipitated with and later probed with anti-FLAG antibodies to detect intact APP. Note that the amount of intact APP is similar in cells transfected with APP alone or in cells cotransfected with APP and caveolin-3 (compare lanes 1, 2). D, Top, The amount of Aβ-amyloid secreted into the medium was measured with a double-sandwich ELISA that uses the antibodies 4G8 and 6E10. Note that a significant reduction of Aβ-amyloid secretion was observed in cells cotransfected with APP and caveolin-3 (p < 0.05) compared with that in cells transfected with APP alone.Bottom, A double-sandwich ELISA used the antibodies BAN50 and BA27 or antibodies BAN50 and BC05 to measure separately the amounts of Aβ40 and Aβ42, respectively. Note that the ratio of Aβ1-42/(Aβ1-40 + Aβ1-42) was not significantly altered by cotransfection of APP with caveolin-3. E, Schematic diagram of caveolin-3 and APP cDNAs is shown.