Fig. 2

Comparative analysis of induced dimerization of APLP1 and APLP2 with cysteine mutants. A, B Schematic illustration of the used cysteine mutants for APLP1 and APLP2, C APLP1 WT, APLP1 R579C and APLP1 E580C were heterologously expressed in N2a cells. Non-transfected N2a cells served as a negative control. Equal amounts of cell lysates were prepared in samples with or without DTT, to allow visualization of cysteine dimers via disulfide bridges. Separation on denaturing SDS-gels followed and Western blot detection with an α-HA antibody. D APLP1 WT, APLP1 R579C, APLP1 E580C, and APLP1 F1 were heterologously expressed in N2a cells. APLP1 F1 expressing cells were either treated with 100 nM of the rapamycin analogue AP20187 overnight to induce dimerization of APLP1 or with the solvent ethanol as a negative control. Non transfected N2a cells served as a further negative control. The samples were analyzed via semi-denaturing Blue Native PAGE and Western blot analysis followed with an α-HA antibody. E APLP2 WT, APLP2 L690C, and APLP2 S692C were transiently transfected in N2a cells. Equal amounts of protein were denatured in sample buffer with and without DTT and analyzed after SDS PAGE via Western blot detection with α-HA antibody. F APLP2 WT, APLP2 L690C, APLP2 S692C, and APLP2 F1 were heterologously expressed in N2a cells. APLP2 F1 expressing cells were either treated with 100 nM of the rapamycin analogue AP20187 overnight to induce dimerization of APLP2 or with the same volume of the vehicle ethanol as a negative control. Non transfected N2a cells served as a further negative control. The samples were analyzed via Blue Native PAGE and Western blot analysis followed with an α-HA antibody