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Results: 6

1.
FIGURE 3.

FIGURE 3. From: Expression of the Anti-amyloidogenic Secretase ADAM10 Is Suppressed by Its 5?-Untranslated Region.

The uORFs are not involved in the translational repression of ADAM10. A, HEK293 cells were transfected with 5′-UTR ADAM10 plasmids bearing the indicated uORF mutations. Representative Western blots for ADAM10, β-actin, and GFP are shown. B, quantification of ADAM10 levels was performed as described for Fig. 1. The results are expressed as the means ± S.D. of at least three independent experiments.

Sven Lammich, et al. J Biol Chem. 2010 May 21;285(21):15753-15760.
2.
FIGURE 5.

FIGURE 5. From: Expression of the Anti-amyloidogenic Secretase ADAM10 Is Suppressed by Its 5?-Untranslated Region.

Effects of uORF mutations and deletions of the ADAM10 5′-UTR on the expression of a Firefly luciferase reporter. HEK293 cells were transfected with firefly luciferase (Luc) cDNA constructs containing the indicated variants of the ADAM10 5′-UTR and Renilla luciferase as a transfection control. Firefly luciferase activity was normalized to Renilla luciferase activity, and the signal for firefly luciferase containing the entire 5′-UTR of ADAM10 was set to 1. The results are expressed as the means ± S.D. of three independent experiments made in triplicate.

Sven Lammich, et al. J Biol Chem. 2010 May 21;285(21):15753-15760.
3.
FIGURE 4.

FIGURE 4. From: Expression of the Anti-amyloidogenic Secretase ADAM10 Is Suppressed by Its 5?-Untranslated Region.

Effects of 5′-UTR deletions on ADAM10 expression. HEK293 cells were transfected with the indicated 5′-UTR ADAM10 cDNA constructs. Representative Western blots for V5-tagged ADAM10 and quantification of ADAM10 signals from cells transfected with ADAM10 cDNA constructs lacking either parts of the 5′ end (A and B) or the 3′ end (C and D) of the 5′-UTR are shown. The values were normalized to β-actin and GFP levels. In both cases the ADAM10 signal for cells transfected with 5′-UTR ADAM10 was set to 1. The results are expressed as the means ± S.D. of at least three (C) or six (D) independent experiments.

Sven Lammich, et al. J Biol Chem. 2010 May 21;285(21):15753-15760.
4.
FIGURE 2.

FIGURE 2. From: Expression of the Anti-amyloidogenic Secretase ADAM10 Is Suppressed by Its 5?-Untranslated Region.

The 5′-UTR of ADAM10 represses translation of a Firefly luciferase reporter. A, HEK293 cells were transfected with firefly luciferase (Luc) containing the 5′-UTR of ADAM10 or with firefly luciferase and with Renilla luciferase as transfection control. Firefly luciferase activity was normalized to Renilla luciferase, and the signal for firefly luciferase containing the 5′-UTR of ADAM10 was set to 1. The results are expressed as the means ± S.D. of three independent experiments made in triplicate. B, equal amounts of in vitro transcribed luciferase mRNAs with and without the 5′-UTR of ADAM10 (upper panel) were subjected to in vitro translation using nuclease-treated rabbit reticulocyte lysate (lower panel).

Sven Lammich, et al. J Biol Chem. 2010 May 21;285(21):15753-15760.
5.
FIGURE 1.

FIGURE 1. From: Expression of the Anti-amyloidogenic Secretase ADAM10 Is Suppressed by Its 5?-Untranslated Region.

The 5′-UTR of ADAM10 represses ADAM10 expression. A, HEK293 cells were transiently transfected with ADAM10 with or without 5′-UTR and GFP as a transfection control. Representative Western blots of V5-tagged ADAM10 (top panel), β-actin (middle panel), and GFP expression (bottom panel) are shown. B, quantification of Western blots shown in A. ADAM10 protein was normalized to GFP (transfection control) and β-actin levels (loading control). The signal for ADAM10 with 5′-UTR was set to 1. The results are expressed as the means ± S.D. of six independent experiments. C, quantification of ADAM10 mRNA by real time reverse transcription-PCR from cells shown in A. ADAM10 mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. The values are the means ± S.D. of at least six different experiments, and the signal for 5′-UTR ADAM10 mRNA is set to 1. D, reduction of ADAM10 expression is not cell type-specific. COS7 cells and SH-SY5Y were transiently transfected with ADAM10 cDNA constructs with or without 5′-UTR and GFP as a transfection control. An asterisk indicates an unspecific band.

Sven Lammich, et al. J Biol Chem. 2010 May 21;285(21):15753-15760.
6.
FIGURE 6.

FIGURE 6. From: Expression of the Anti-amyloidogenic Secretase ADAM10 Is Suppressed by Its 5?-Untranslated Region.

Effect of Δ1–259 5′-UTR ADAM10 on APP processing. A, HEK293 cells stably overexpressing APP were transiently transfected with wild-type 5′-UTR ADAM10, Δ1–259 5′-UTR ADAM10, or empty vector (mock). The cell lysates were analyzed by immunoblotting using the V5 antibody for ADAM10 and antibody 6687 for full-length APP; im, the immature form; m, the mature form of APP and ADAM10. β-Actin was used as a loading control. The conditioned media were analyzed by immunoblotting for APPsα and Aβ using antibody 6E10 and APPsβ using antibody 192wt. B, quantification of APPsα, APPsβ, and Aβ levels from Western blots shown in A. The values for mock transfected cells were set to 100%. The bars represent the means of eight experiments with error bars indicating the S.E. The asterisks indicate statistical significance (one-way analysis of variance with Dunnett's post-test) relative to mock transfected cells (***, p < 0.001). C, sandwich immunoassay of Aβ40 species from conditioned media of cells transfected with the indicated cDNA constructs shown in A. Aβ40 values of mock transfected cells were set to 100%. The bars represent the means of eight experiments with the error bars indicating the S.E. The asterisks indicate statistical significance (one-way analysis of variance with Dunnett's post-test) relative to mock transfected cells (***, p < 0.001).

Sven Lammich, et al. J Biol Chem. 2010 May 21;285(21):15753-15760.

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