S-Palmitoylation-deficient nicastrin and APH-1 assemble γ-secretase with other subunits. A, immunoblot analysis of γ-secretase subunits in knock-out MEF. WT or Cys mutant nicastrin and APH-1 were stably co-expressed by retroviral transduction in NCT–/– and APH-1ac–/– MEF, respectively. For control, MEFs were transduced with empty retroviral vector (Vec). NCT–/– MEF were also sequentially transduced with nicastrin and APH-1 to generate double WT (dWt) and double mutant (dMut) pools. Immunoblots were performed to monitor transgene expression, as well as rescue stability and endoproteolytic processing of PS1, maturation of nicastrin, and stabilization of PEN2. B, acyl-biotin exchange labeling of nicastrin and APH-1 in NCT–/– dWt and dMut MEF, as described in the legend to . C, co-immunoprecipitation analysis of APH-1 replacement. NCT–/– dWt and dMut MEF were lysed in a buffer containing 1% CHAPSO and immunoprecipitated with PS1NT or αPS1Loop antibodies. The blots were sequentially probed with polyclonal APH-1 antibody, A1tag, and PS1NT or αPS1Loop antibodies. D, co-immunoprecipitation analysis of PS1 complexes. NCT–/– dWt and dMut MEF were lysed in 1% CHAPSO buffer and PS1 association with nicastrin, APH-1, and PEN-2 were analyzed by co-immunoprecipitation using αPS1Loop antibody, at steady state (control) and 8 h after translation arrest in the presence of cycloheximide (CHX). The blots were sequentially probed with antibodies against each γ-secretase subunit (SP716, 9E10, PNT2, and PS1NT). Note that the signals for PEN-2 and PS1 CTF are much stronger in PS1 co-immunoprecipitates (IP) compared with 4% input lanes, where they are detectible only upon longer exposure of the blots to films (not shown).