|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. Fe65 transports AICD to nuclei where it can regulate transcription. (A) Blocking nuclear export with leptomycin B for 9 hours leads to an accumulation of Fe65 and AICD in nuclei. In cells co-expressing Fe65 and AICD, leptomycin B caused the formation of nuclear AICD-containing spots that were never observed in cells without the cotransfection of Fe65, or in the absence of leptomycin B (compare Fig. 1A,B). Bar, 10 µm. (B) Leptomycin B (24 hours) increased fluorescence signal intensity ratios of nuclear versus extranuclear staining for AICD and Fe65 in co-expressing cells. Without the expression of Fe65, leptomycin B treatment did not change the nuclear/extranuclear signal ratio for AICD. (C) Western blots of nuclear (n) and post-nuclear supernatant (PNS) fractions from AICD- and Fe65-expressing cells treated with lepomycin B (0 to 24 hours) showed increased nuclear retention of Fe65. The nuclear/PNS signal ratio for Fe65 increased to 247% after 9 hours and to 527% after 24 hours, as compared with a 100% baseline. Marker proteins are 19, 28, 39, 51, 64, 97 and 191 kDa. (D,E) Real-time PCR of clonal cell lines with and without the induced expression of citrine-AICD revealed that AICD increased the expression of APP, BACE and Tip60 (Mann-Whitney U, P<0.05). AICD did not change the expression of Fe65, Tip60, ADAM10, PSEN1, or the Notch-effector gene Hes1. The AICD-induced expression of KAI1 and Gsk3ß was confirmed. All values were normalized against GAPDH expression levels and expressed as fold baseline without induction of AICD expression. Data represent means ± s.e.m. of five experiments, each done in triplicate. (F,G) Clonal HEK293 grown for 48 hours without (F) or with (G) the induction of AICD-citrine expression. 6E10-staining for endogenous APP reveals increased protein levels induced by AICD. Bar, 20 µm. (H) Whole cell extracts of naive HEK293 and AICD-expressing clonal cell lines. Western blots were analyzed with antibodies against the N- or C-terminus of APP, against GFP to detect the citrine-AICD fusion protein and GAPDH as a loading control. Naive HEK293 (C, lane 1) were also treated with the 
-secretase inhibitor DAPT, which leads to accumulation of 
- and ß-stubs (DAPT, lane 2). Clonal HEK293 were induced for 3, 6 or 48 hours (lane 4 to 6) to express citrine-AICD. Bar diagram shows densitometric analysis of full-length APP and -together with ß-stubs from three independent experiments. Relative protein levels are determined with respect to uninduced cells (0 hrs).
|
