Fig. 3

The extent of APP, APLP1, APLP2 cis-dimers and trans-dimers in N2a cells. A Experimental design of the cis-co-immunoprecipitation. B Cis-co-immunoprecipitation of APP, APLP1, and APLP2 (cis-homodimers). N2a cells were transiently co-transfected with APP c-myc and APP HA, APLP1 c-myc and APLP1 HA or APLP2 c-myc and APLP2 HA constructs. Co-transfection with an empty vector served as a negative control. Equal amounts of cell lysates were loaded on an SDS gel and analyzed via Western blot with primary α-HA or α-c-myc antibodies (input controls). Further, equal amounts of cell lysates were used for immunoprecipitation with α-HA coated agarose beads. The samples were separated on an SDS gel and subjected for Western blot detection with primary antibody α-c-myc to detect the co-immunoprecipitated proteins. The same membrane was incubated afterwards with α-HA antibody to visualize total amounts of immunoprecipitated proteins. C Quantification of data shown in A. Bars represent mean values ± SEM; n = 3, unpaired Student’s t test *p < 0.05, **p < 0.01, ***p < 0.001. D Experimental design of the trans-co-immunoprecipitation. E Trans-co-immunoprecipitation of APP, APLP1 or APLP2 (trans-homodimers). N2a cells were transiently transfected in separate dishes with APP, APLP1 or APLP2 HA or c-myc tagged constructs. Further, transfections of APP myc or empty vector served as a negative control. 4 h post-transfection, the transfected cells of two dishes were combined: APP myc and APP HA, APLP1 myc and APLP1 HA, APLP2 myc and APLP2 HA. Equal amounts of cell lysates were loaded on an SDS gel and analyzed via Western blot with primary α-HA or α-c-myc antibodies (input controls). Further, equal amounts of cell lysates were used for immunoprecipitation with α-HA antibody coated agarose beads. The samples were separated on an SDS gel and subjected for Western blot detection with the primary antibody α-c-myc to detect the co-immunoprecipitated proteins. The same membrane was incubated afterwards with an α-HA antibody to detect total amounts of immunoprecipitated proteins