A: HA-tagged GGA3DN, HA-tagged GGA3 w.t. or vector alone with myc-tagged BACE in H4-APP751 cells. WB analysis performed with anti-HA antibody (Cell Signaling) revealed the expression of GGA3 construct. WB analysis performed with anti-myc (Cell Signaling) revealed that BACE levels were increased in the cells expressing GGA3DN compared to cells expressing vector alone or GGA3. B: The graph represents mean ± SEM of at least 6 BACE levels measurements. Densitometry was performed using Versadoc Imager and QuantityOne software (Bio-Rad). BACE densitometry values were normalized against GAPDH values. Unpaired T-test with Welch correction was used for statistical analysis. p = 0.0014 vector vs GGA3DN. p = 0.0008 GGA3 w.t vs GGA#DN. C: The graph represents mean ± SEM of at least 6 Aβ0 measurements by ELISA. Aβ concentration was normalized against the concentration of protein in cell lysates.

A: Northern Blot analysis: H4-APP751 cells were treated with STS during time course experiments. Total RNA was extracted, and 20 μg of the resulting RNA were analyzed by northern blotting with BACE cDNA. Ethidium staining of the gel (lower panel) confirmed equal RNA loading and absence of non-specific degradation. B: Pulse-chase analysis. H4-APP751 cells were transiently transfected with BACE-Myc cDNA and were metabolically labeled after 24 hr. Lysates from each time point were immunoprecipitated with anti-Myc antibody. C: Protein amounts were quantified by phosphorimager and represented in the graph. The mean ± SEM of at least three experiments is shown. D-E-F: For the TACE and APP pulse-chase, H4-APP751 cells were metabolically labeled. Lysates from each time point were immunoprecipitated wither with anti-TACE or anti-APP (A8717) antibody. Protein amounts were quantified by phosphorimager and represented in the graph. The mean ± SEM of at least three experiments is shown. D: Cycloheximide degradation time-course: BACE, TACE, and APP proteins were detected by Western blot at various times after addition of CHX (40 μg/ml) only or STS + CHX in H4-APP751 cells. The degradation of BACE was decreased while the degradation of TACE and APP was unchanged during the apoptosis.

A: Multiple sequence alignment of human (NP_619525), mus musculus (NP_766636), Bos taurus (XP_587687) and canis familiaris (XP_540429) GGA3. Putative caspase consensus sequence in the hinge and GAE domain are in box. The red bar indicates the epitope recognized by the anti GGA3 antibody. The Hinge domain starts at D313 and ends at V599. The yellow and pink background indicates GAT and GAE domain, respectively. B: Site-directed mutagenesis of D313 to alanine prevents the caspase 3-mediated cleavage of in vitro translated GGA3 (cold) at one of the three major sites (WB with anti-GGA3 Antibody). C: H4 cells were transiently transfected with empty vector, GGA3 wild type (w.t.), and GGA3D313A/D328A/D333A/D428A. The overexpression of GGA3 w.t. induced an artefactual caspase-mediated cleavage (stars indicate the fragments). The cleavage was inhibited when cells were treated with zVAD during the transfection. The caspase-derived fragments were not detected in the GGA3D313A/D328A/D333A/D428A when cells where treated with STS. However zVAD treatment, but not GGA3D313A/D328A/D333A/D428A, preserved full-length GGA3.

A: H4-APP751 cells were transfected with 200 nM siRNA GGA3 or 200 nM siNeg control. After 72 hr, GGA3 protein levels were determined by WB with anti-GGA3 antibody (Transduction Laboratories). GAPDH was used as loading control. B: After 72 hr, a sister plate of the same cells was co-transfected with myc-tagged BACE and siGGA3 or siNeg control. EGFR levels detected by WB with anti-EGFR antibody (Cell Signaling) were increased in the siGGA3 treated cells. BACE protein levels were detected by WB using anti-myc polyclonal antibody (Cell signaling) were also increased in the siGGA3 treated cells. Levels of GAPDH were unchanged. C-D: the graph represents mean ± SEM of 6 or 7 BACE levels measurements for H4-APP751 and H4 APPSwe, respectively. Densitometry was performed using Versadoc Imager and QuantityOne software (Bio-Rad). BACE densitometry values were normalized against GAPDH values. Unpaired T-test with Welch correction was used for statistical analysis. E: Full-length APP and APP-C99 levels were detected by WB with WO2 antibody in H4-APPSWE cells. F-G: the graph represents mean ± SEM of 6 or 9 Aβ0 measurements by ELISA in H4-APP751 or H4 APPSWE, respectively. Aβ concentration was normalized against the concentration of protein in cell lysates. Unpaired T-test with Welch correction was used for statistical analysis.

A: Cerebral ischemia was induced by middle cerebral artery occlusion for 1 hr in female rats. After desired times of reperfusion, the animals were sacrificed. The brains were dissected in the ischemic (ipsilateral, I) and not-ischemic (contralateral, C) hemispheres and each hemisphere in cortex (Ctx) and sub-cortex (Sub-Ctx). A-B: WB analysis with anti-BACE antibody (ABR) revealed an increase in BACE proteins levels in the ischemic hemisphere (samples were run in duplicate) after 48 hr of reperfusion. β-tubulin was used as loading control. The densitometry analysis of BACE after normalization against β-tubulin levels is represented in the graph. Mean ± SEM of 5 rats. Statistical analysis was performed using Mann-Whitney test. WB with an anti-APP-CTF antibody (A8717, Sigma) revealed an APP caspase-derived fragment in the rat ischemic hemisphere (samples were run in duplicate) after 48hr of reperfusion. C: WB analysis with anti-GGA3 antibody revealed a decrease in GGA3 full-length and a longer exposure two fragments of GGA3 generated during ischemia after 12 and 48 hrs of reperfusion (C=contralateral hemisphere; I=ipsilateral hemisphere; SubCtx= subcortex; Ctx=cortex).

A. Western Blot analysis of temporal cortex of human brains. AD= Alzheimer's disease. ND= non-demented control. BACE was detected by SECB1. GGA3 was detected by anti-GGA3 antibody. GAPDH was used as loading control. B-C. BACE and GGA3 densitometry values were normalized against GAPDH values. At least triplicate of each samples were analyzed. The graphs represent mean ± SEM of 19 ND and 20 AD. Unpaired t-test and unpaired t-test with Welch correction ware used to perform statistical analysis of BACE and GGA3 levels, respectively. D-E. Linear correlation analysis between BACE and GGA3 levels in AD and ND, respectively. The dotted line indicates the 95% confidence interval. F. GGA3 mRNA was quantified by real time PCR. The graph represents mean ± SEM of 10 ND and 10 AD. SQ=starting quantity. Please note that the levels of GGA3 protein were significantly decreased in the same samples. G. Western Blot analysis of temporal cortex of human brains. AD= Alzheimer's disease. ND= non-demented control. Full-length caspase 3 was detected with anti-caspase 3 antibody. To show that full-length caspase 3 decrease during apoptosis, lysates from control (C), treated with (STS), and treated with STS+zVAD H4 cells were also included. GAPDH was used as loading control.

A: Apoptosis was induced in H4-APP751 cells by STS treatment during time course experiments. Western blot analysis with an anti-GGA3 antibody revealed cleavage of GGA3 in two major fragments of ∼48 and ∼37 kDa (indicated by the arrows) in apoptotic H4 cells, which was prevented by caspase inhibition (zVAD). A longer exposure (Long exp) better evidentiates the caspase-derived fragments. Western Blot analysis with anti-β-catenin antibody revealed that β-catenin was cleaved by caspase (casp-fragment) in the H4-APP751 cells with a temporal pattern similar to that observed for GGA3. B: recombinant caspase 3 cleaves in vitro translated GGA3 (labeled with [³⁵S] methionine) in several fragments. The stars indicate the fragments detected by WB in Fig. 3A. The additional fragments most likely are caspase-derived N-terminal fragments (NTFca). C: recombinant caspase 3 cleaves in vitro translated GGA3 (cold methionine) in three fragments detected in WB by anti-GGA3 antibody (stars indicate the fragments). D: recombinant caspase 3 cleaves endogenous GGA3 from lysates of control cells with a pattern similar to the one observed in H4 apoptotic lysates (stars indicate the fragments). E: apoptosis induced in H4 cells stably overexpressing GGA3 produces a cleavage of GGA3 identical to the one produced by recombinant caspase 3 (stars indicate the fragments). Non-specific bands are also detected just below the ∼48 kDa fragment and in correspondence of the ∼37 kDa fragment (N.S.). F: Schematic representation of GGA3 caspase cleavage.

A: Schematic representation of APP caspase sites, and antibody recognition sites. B: Apoptosis was induced in human H4 neuroglioma cells expressing APP751 by STS treatment. Western blot analysis performed with WO2 antibody revealed APP-C99 and APP-C99ΔC31 during time-course experiments. Western blot analysis with anti-caspase 3 active fragment antibody showed caspase 3 activation as early as 6 hr during STS treatment. Limited caspase activity and a small increase of APP-C99 in the untreated cells at time point 12 (horizontally compressed band) and 24 hr was also detected in the control sample owing to the fact that the control cells were grown in serum-free media. C: WB analysis with an antibody, ASP-1 (Oncogene), that recognizes only the first aspartyl residue of Aβ region confirmed that the 12kDa APP fragment increasing following caspase activation is APP-C99 in the cells H4-APP751, and also in human SH-SY5Y and murine N2A cells expressing only endogenous APP. D-F: Western blot analysis with anti-BACE antibody revealed increased BACE protein levels following apoptosis induced by STS or etoposide treatment, respectively. Cu,Zn-SOD or β-tubulin were used as a loading control. Densitometry analysis was performed using NIH image software. The graphs represent BACE levels expressed as percentage increase versus BACE levels in control cells (100%). Each bar represents the mean ± SEM of at least three experiments.