Figure 1.

(A) Notch1 is coimmunoprecipitated with PS1. 293 cells were transiently transfected with (+) or without (−) FLN6mt and PS1 as indicated. Cells were lysed in SDS buffer and analyzed directly (crude lysate), or in coimmunoprecipitation (IP) lysis buffer and immunoprecipitated with the indicated antibody or with normal rabbit serum. Precipitated proteins were resolved by SDS/PAGE and immunoblotted with α-myc, which detects the myc epitope tag at the C terminus of FLN6mt. Crude lysates equal 20% of the protein used in coimmunoprecipitations. Bands migrating at ≈64 kDa (lane 1) and ≈95 kDa (lanes 7 and 8) below TMIC-N are apparently degradation products that occur postlysis in some experiments and not specific products of cellular metabolism. Migration of molecular weight markers (in kDa) is shown on right. (B) Notch and PS1 do not associate postlysis. HEK 293 cells were transfected with FL-N with an HA epitope tag at the C terminus and full-length human PS bearing a C-terminal myc epitope as indicated (+ or −). In lane 1, cells were lysed in SDS and analyzed directly. In lane 2, cultures, one expressing Notch-HA (A) and one expressing PS1-myc (B), were mixed immediately following lysis with coimmunoprecipitation lysis buffer and incubated overnight. Lysates were immunoprecipitated with anti-myc as under coimmunoprecipitation conditions. Lane 3 is the positive control in which both proteins were coexpressed in the same culture before lysis. Immunoblot was with polyclonal anti-HA; migration of molecular weight markers (in kDa) is shown on the left.