Caveolin-3 upregulation activates beta-secretase-mediated cleavage of the amyloid precursor protein in Alzheimer's disease - PubMed (original) (raw)

Fig. 3.

Caveolin-3 physically interacts with APP and the presenilins in human brain, primary cultures of astrocytes, CRT astrocytoma cells, and COS-7 cells. A, Human brain. Extracts were prepared and immunoprecipitated with a caveolin-3–specific antibody (left,center) or with an NR1-specific antibody (right). Left, Center, After SDS-PAGE and transfer to nitrocellulose, these immunoprecipitates were probed by immunoblot analysis with the following antibodies: 4G8 to detect APP [left, top; lane 2 (in this and subsequent figures, lane 1 is the_left-hand lane_)], anti-presenilin-1 (-PS-1) directed against the N-terminal fragment of presenilin-1 (center, top; lane 2), anti-PS-2 (1209) directed against the N-terminal fragment of presenilin-2 (center,second from top; lane 2), and anti-tubulin (center, third from_top_; lane 2). Immunoprecipitation with normal mouse IgG (NMG) was performed in parallel as a negative control and did not show any association with APP, PS-1, or PS-2, as expected (all; lane 1). Tubulin was clearly detected in the starting material by immunoblot analysis with anti-tubulin antibody (center,bottom). Right, In the NR1 immunoprecipitates, NR1 was clearly detected by immunoblot analysis with NR1 antibody (top); however no APP, PS-1, PS-2, or caveolin-3 was detected by immunoblot analysis (middle,bottom). B, Primary cultured astrocytes. Extracts were prepared and immunoprecipitated with a caveolin-3–specific monoclonal antibody (bottom;lane 2). After SDS-PAGE and transfer to nitrocellulose, these immunoprecipitates were probed by immunoblot analysis with 4G8 to detect APP (top; lane 2). Immunoprecipitation with NMG was performed in parallel as a negative control and did not show any association with APP, as expected (lane 1). C, CRT astrocytoma cell line. Extracts were prepared and immunoprecipitated with a caveolin-3–specific monoclonal antibody (left,center) or with an NR1-specific monoclonal antibody (right). Left, Center, After SDS-PAGE and transfer to nitrocellulose, these immunoprecipitates were probed by immunoblot analysis with the following antibodies: 4G8 to detect APP (left, top; lane 2), anti-PS-1 directed against the N-terminal fragment of presenilin-1 (center,top; lane 2), anti-PS-2 directed against the N-terminal fragment of presenilin-2 (center, second from_top_; lane 2), and anti-tubulin (center, third from top;lane 2). Immunoprecipitation with NMG was performed in parallel as a negative control and did not show any association with APP, PS-1, or PS-2, as expected (all;lane 1). Tubulin was clearly detected in the starting material by immunoblot analysis with anti-tubulin antibody (center, bottom). Right, In the NR1 immunoprecipitates, NR1 was clearly detected by immunoblot analysis with NR1 antibody (top); however no APP, PS-1, PS-2, or caveolin-3 was detected by immunoblot analysis (middle, bottom). D, COS-7 cells. Cells were cotransfected with HA-tagged APP and myc-tagged caveolin-3 and analyzed by immunoprecipitation and Western blotting. Cells were immunoprecipitated with anti-myc IgG (mAb 9E10), and these immunoprecipitates were analyzed by Western blotting with antibodies directed against HA (to detect APP; top) and myc (to detect caveolin-3; bottom). Under these conditions, immunoprecipitation of caveolin-3 shows that APP coprecipitates. Immunoprecipitation with NMG was performed in parallel as a negative control and showed little or no association with APP.cav-3, Caveolin-3; IP, immunoprecipitation; pAb, polyclonal antibody;MM, molecular marker; N-TF, N-terminal fragment.