Characterization of the anti-presenilin 2 (PS2) antiserum (NT14) by Western blot analysis. (A) Purified bacterially produced proteins or total cellular proteins were sized by SDS-PAGE, transferred to (PVDF) membrane, and probed with affinity-purified NT14. The following were loaded onto the gel: 50 µg total protein extracted from (L/PS1) cells (lane 1), (L/PS2) cells (lanes 2 and 7), mouse N2a neuroblastoma cells (lane 3), N2a cells overexpressing human PS2 (N2a/PS2, lanes 4, 8 and 10) (23), and 200 µg of a membrane pellet prepared from the prefrontal cortex of two control individuals (lanes 5, 6, 9 and 11). The membrane containing lanes 7 and 9 was probed with NT14 binding solution containing glutathione S-transferase fusion protein construct (GST-PS2/NT); lanes 10 and 11, with NT14 binding solution contain GST-PS1/NT (see “Materials and Methods” and Fig. 1). (B) A membrane identical to that used in (A) was probed with an affinity-purified polyclonal antibody (Ab) raised against the large loop domain of PS2 (23). (C) Mouse brain homogenates were prepared from non-transgenic control (lanes 1 and 2) or human PS2 overexpressing transgenic (lanes 3 and 4) mice and analyzed by Western blot. 100 µg of protein was loaded per lane. Lanes 1 and 2 were probed with affinity-purified NT14 and lanes 3 and 4 with the PS2 anti-loop Ab. (D) Total brain extract prepared from human prefrontal cortex (lanes 1 and 3) or a non-transgenic mouse (lanes 2 and 4) was either heated to 56°C (lanes 1 and 2) or placed in a boiling water bath (lanes 3 and 4) for 10 min prior to resolving on SDS-PAGE, transfer to membrane, and probing with NT14. (E and F) Specificity of PS2 detection in human brain. Serial sections from human prefrontal cortex were processed for immunohistochemistry as described in “Materials and Methods” and incubated with either NT14 (D) or NT14 in the presence of GST-PS2/NT (E). L/PS1, L cell expressing human PS1; L/PS2, L cell expessing human PS2; N2a; Neuro-2a; PFC, prefrontal cortex; PS2 tg, PS2 transgenic; non tg, non-transgenic.