TSPAN12 and 17 are required for A10 and γ-secretase association and contribute to the α–β-secretase activity regulation by γ inhibition. (A) 7W cells were transfected with siRNA targeting TSPAN5 + 14, TSPAN12 + 17, or the control. CM were analyzed for APPs-α and lysates for A10 and PS1. (B) S20 cells were transfected with siRNA as in A, and the resultant lysates were immunoprecipitated for γ-secretase by pull-down of Flag–Pen-2 (via an M2 resin). The resulting immunoprecipitates were probed for the coIP of A10 and other γ components. (C) 7W cells were transfected with control siRNA or siRNA targeting both TSPAN12 and 17 and treated in duplicate with increasing amounts of DAPT (3 nM–1 µM). The resulting CM was probed for APPs-α and lysates for A10, PS1, and APP CTFs. (D) CM from C was analyzed by an MSD ELISA for APPs-α and APPs-β. Data are represented as a ratio of APPs-α/APPs-β. A two-way ANOVA with a Sidak’s posttest was performed. *, P < 0.05; ***, P < 0.001. n = 6. CON, control; im, immature; m, mature.