Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: Antibodies information .

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: BACE1 and Hsc70 in AD-affected human brains and in lipid rafts from triple-transgenic Alzheimer’s disease mouse brains. (A) β-amyloid, PHF-1, BACE1 and Hsc70 immunoreactivity in the temporal gyrus of AD-affected brains (AD) and control brains. 40× magnification; scale bar: 20 μm. n = 4. (B) BACE1, Hsc70 and CHIP Western blotting from the temporal gyrus of AD brains (AD); normal human brains were used as controls. A representative plot is shown. βIII-tubulin was used as a loading control. Densitometric quantification was performed; RU = relative units. n = 4, * p < 0.05. (C) Western blotting of BACE1, Hsc70, Hsp90, CHIP and PHF-1 proteins in lipid rafts isolated from the cerebral cortex of 15-month-old wild-type and 3xTg-AD mice. Flotillin-1 was used as a loading control. Densitometric quantification was performed; RU = relative units. n = 3, * p < 0.05. (D) Western blotting of Hsc70 in the cytoplasmic fraction from the cerebral cortex of wild-type and 3xTg-AD mice. RU = relative units. n = 3, * p < 0.05. Representative blots are shown. The arrow shows the band corresponding to the expected molecular weight of the BACE1 protein.

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques: Transgenic Assay, Western Blot, Isolation, Molecular Weight

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: BACE1miR decreases HSF-1, upregulates Hsc70 and induces differential cellular distribution of Hsc70 and Hsp90 in the hippocampus of 3xTg-AD mice. (A) Hippocampi of 18-month-old 3xTg-AD mice at 12 months after injection with AAV2/5-BACE1miR (BACE1miR) or AAV2/5-GFP (GFP). Immunofluorescence of Hsc70 in the CA1 region of 3xTg-AD mice. The white arrowheads show a significant increase in Hsc70 protein expression in the CA1 region that was injected with AAV2/5.BACE1miR compared to the left hemisphere (not injected) and to a right hemisphere injected with AAV2/5-GFP (control). Red: Alexa 594. 10X magnification, scale bar: 20 μm. n = 3. (B) Graph of Hsc70 fluorescence intensity (red channel) in the CA1 area showing a significant increase of Hsc70 expression in BACE1miR-treated mice compared to the left hemisphere (not injected) and right hemisphere injected with AAV2/5-GFP (control). Quantification was performed using image Scope Pro software (Media Cybernetics); RU = relative units. n = 3, * p < 0.05. (C) Cellular fractions from the hippocampi of 15-month-old 3xTg-AD mice that were treated with AAV2/5-BACE1miR (BACE1miR) or AAV2/5-GFP (GFP) for three weeks. Hsc70, CHIP and Hsp90 levels in the (C) cytoplasm, (D) membrane and (E) nuclear fractions. (F) HSF-1 protein expression in the hippocampi of 15-month-old 3xTg-AD mice at three weeks after injection with AAV2/5-BACE1miR (BACE1miR) or AAV2/5-GFP (GFP). Representative blots are shown. βIII tubulin, flotillin and NeuN were used as loading controls or as fraction controls. Densitometric quantification was performed; RU = relative units. n = 6, * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques: Injection, Immunofluorescence, Expressing, Fluorescence, Software

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: BACE1miR decreased hyper-phosphorylated tau in a proteosome-independent manner in primary neuronal cultures. (A) BACE1, PHF-1, Hsp90, Hsc70, CHIP, pAkt (ser473), GSK3 (pSer9) and CDK5 Western blots from primary neuronal cultures transduced at DIV5 (7 days transduction) with AAV2/5-BACE1miR or AAV2/5-GFP as a control. Representative blots are shown. Actin was used as a loading control. Densitometric quantification was performed; RU = relative units. n = 4, * p < 0.05, * p < 0.001. (B) Western blotting of primary cortical cultures transduced with AAV2/5-BACE1miR or AAV2/5-GFP at DIV5 for 7 days. Neurons were exposed to lactacystin (synthetic; 10 μM), KNK437 heat shock protein inhibitor I, (100 μM) or DMSO for 24 h. Representative blots of (B) PHF-1, (C) Hsc70, (D) Hsp90, (E) CHIP and (F) LC3-B. βIII-tubulin was used as a loading control. Densitometric quantification was performed; RU = relative units. n = 4, * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques: Western Blot, Transduction

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: BACE1 silencing upregulates Hsc70 and LAMP-2. Hippocampi of 15-month-old 3xTg-AD mice were analyzed at three weeks after injection with AAV2/5-BACE1miR (BACE1miR) or AAV2/5-GFP (GFP). (A) LC3-B protein levels in the membrane and cytoplasm fractions. (B) BACE1, Hsc70, and LAMP-2A protein levels in lipid rafts and LAMP2-A in the cytoplasm from hippocampi of 15-month-old 3xTg-AD mice treated for three weeks with AAV2/5-BACE1miR (BACE1miR) or AAV2/5-GFP (GFP). βIII tubulin and flotillin were used as a loading control and a fraction control respectively Densitometric quantification was performed; RU = relative units. n = 4, * p < 0.05.

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques: Injection

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: Silencing of BACE1 upregulates the survival signaling in 3xTg-AD mice. Survival and quality control regulation pathways were analyzed. (A) Bcl2/Beclin-1, (B) Akt/FoxO3, (C) Hsc70/Hsp90/CHIP protein levels, (D) mTOR pathway and its activity (E) from the hippocampi of 3xTg-AD mice 24-month-old after 6 months of injection with AAV2/5-BACE1miR and AAV2/5-GFP version as control. Representative blots are shown. RU = relative unit. n = 4, * p < 0.05.

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques: Activity Assay, Injection

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: Antibodies information .

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: BACE1 and Hsc70 in AD-affected human brains and in lipid rafts from triple-transgenic Alzheimer’s disease mouse brains. (A) β-amyloid, PHF-1, BACE1 and Hsc70 immunoreactivity in the temporal gyrus of AD-affected brains (AD) and control brains. 40× magnification; scale bar: 20 μm. n = 4. (B) BACE1, Hsc70 and CHIP Western blotting from the temporal gyrus of AD brains (AD); normal human brains were used as controls. A representative plot is shown. βIII-tubulin was used as a loading control. Densitometric quantification was performed; RU = relative units. n = 4, * p < 0.05. (C) Western blotting of BACE1, Hsc70, Hsp90, CHIP and PHF-1 proteins in lipid rafts isolated from the cerebral cortex of 15-month-old wild-type and 3xTg-AD mice. Flotillin-1 was used as a loading control. Densitometric quantification was performed; RU = relative units. n = 3, * p < 0.05. (D) Western blotting of Hsc70 in the cytoplasmic fraction from the cerebral cortex of wild-type and 3xTg-AD mice. RU = relative units. n = 3, * p < 0.05. Representative blots are shown. The arrow shows the band corresponding to the expected molecular weight of the BACE1 protein.

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques: Transgenic Assay, Western Blot, Isolation, Molecular Weight

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: BACE1miR decreases HSF-1, upregulates Hsc70 and induces differential cellular distribution of Hsc70 and Hsp90 in the hippocampus of 3xTg-AD mice. (A) Hippocampi of 18-month-old 3xTg-AD mice at 12 months after injection with AAV2/5-BACE1miR (BACE1miR) or AAV2/5-GFP (GFP). Immunofluorescence of Hsc70 in the CA1 region of 3xTg-AD mice. The white arrowheads show a significant increase in Hsc70 protein expression in the CA1 region that was injected with AAV2/5.BACE1miR compared to the left hemisphere (not injected) and to a right hemisphere injected with AAV2/5-GFP (control). Red: Alexa 594. 10X magnification, scale bar: 20 μm. n = 3. (B) Graph of Hsc70 fluorescence intensity (red channel) in the CA1 area showing a significant increase of Hsc70 expression in BACE1miR-treated mice compared to the left hemisphere (not injected) and right hemisphere injected with AAV2/5-GFP (control). Quantification was performed using image Scope Pro software (Media Cybernetics); RU = relative units. n = 3, * p < 0.05. (C) Cellular fractions from the hippocampi of 15-month-old 3xTg-AD mice that were treated with AAV2/5-BACE1miR (BACE1miR) or AAV2/5-GFP (GFP) for three weeks. Hsc70, CHIP and Hsp90 levels in the (C) cytoplasm, (D) membrane and (E) nuclear fractions. (F) HSF-1 protein expression in the hippocampi of 15-month-old 3xTg-AD mice at three weeks after injection with AAV2/5-BACE1miR (BACE1miR) or AAV2/5-GFP (GFP). Representative blots are shown. βIII tubulin, flotillin and NeuN were used as loading controls or as fraction controls. Densitometric quantification was performed; RU = relative units. n = 6, * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques: Injection, Immunofluorescence, Expressing, Fluorescence, Software

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: BACE1miR decreased hyper-phosphorylated tau in a proteosome-independent manner in primary neuronal cultures. (A) BACE1, PHF-1, Hsp90, Hsc70, CHIP, pAkt (ser473), GSK3 (pSer9) and CDK5 Western blots from primary neuronal cultures transduced at DIV5 (7 days transduction) with AAV2/5-BACE1miR or AAV2/5-GFP as a control. Representative blots are shown. Actin was used as a loading control. Densitometric quantification was performed; RU = relative units. n = 4, * p < 0.05, * p < 0.001. (B) Western blotting of primary cortical cultures transduced with AAV2/5-BACE1miR or AAV2/5-GFP at DIV5 for 7 days. Neurons were exposed to lactacystin (synthetic; 10 μM), KNK437 heat shock protein inhibitor I, (100 μM) or DMSO for 24 h. Representative blots of (B) PHF-1, (C) Hsc70, (D) Hsp90, (E) CHIP and (F) LC3-B. βIII-tubulin was used as a loading control. Densitometric quantification was performed; RU = relative units. n = 4, * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques: Western Blot, Transduction

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: Silencing of BACE1 upregulates the survival signaling in 3xTg-AD mice. Survival and quality control regulation pathways were analyzed. (A) Bcl2/Beclin-1, (B) Akt/FoxO3, (C) Hsc70/Hsp90/CHIP protein levels, (D) mTOR pathway and its activity (E) from the hippocampi of 3xTg-AD mice 24-month-old after 6 months of injection with AAV2/5-BACE1miR and AAV2/5-GFP version as control. Representative blots are shown. RU = relative unit. n = 4, * p < 0.05.

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques: Activity Assay, Injection

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: Antibodies information .

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: β-Secretase 1’s Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice

doi: 10.3389/fncel.2015.00498

Figure Lengend Snippet: Antibodies information .

Article Snippet: The membranes were incubated overnight at 4°C with rabbit anti-BACE1 C-terminal (485–501; 1:250, Calbiochem), rabbit anti-BACE2 (Ab2; 44–59; 1:500, Calbiochem), rabbit anti-Hsc70 (Hsp73; 1:1000, Assay Designs), mouse anti-Hsp90 (AC88; 1:500, Assay Designs), rabbit anti-CDK5 (C-8; 1:1000, Santa Cruz Biotechnology), mouse anti-human-PHF-tau (1:500, Pierce Biotechnology), rabbit anti-phospho-GSK-3β (Ser9; 1:1000, Cell Signaling Technology), rabbit anti-phospho-Akt (Ser473; 1:1000, Cell Signaling Technology), rabbit anti-CHIP (N-terminal; 1:1000, Sigma-Aldrich), rabbit anti-LC3B (1:500, Cell Signaling Technology), rabbit anti-LAMP2-A (1:1000, Sigma-Aldrich), mouse anti-flotillin-1 (1:1000, BD Biosciences), rabbit anti-mTOR (1:1000, Cell Signaling Technology), mouse anti-Bcl-2 (1:500, Santa Cruz Biotechnology), rabbit anti-HSF1 (1:1000, Cell Signaling Technology), rabbit anti-phospho-mTOR (Ser2448; 1:1000, Cell Signaling Technology), rabbit anti-phospho-p70 S6 kinase (Thr389; 1:1000, Cell Signaling Technology), rabbit anti-beclin-1 (1:1000, Cell Signaling Technology), rabbit anti-FoxO3 (1:1000, Cell Signaling Technology), mouse anti-presenilin-1 (APS 18; 1:250, Pierce Biotechnology), PHF-1 monoclonal antibody, which recognizes TaupSer-396/404 (1:1000) donated by P. Davies (Feinstein Institute for Medical Research, Manhasset, NY, USA), mouse anti-Phospho-PHF-tau pSer202/Thr205 Antibody (AT8; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr212/Ser214 Antibody (AT100; 1:1000, Thermo Fisher Scientific), mouse anti-phospho-PHF-tau pThr231 Antibody (AT180), (1:1000, Thermo Fisher Scientific), mouse anti-Tau Antibody (TAU-5; 1:1000, Invitrogen) anti-APP A4 (Millipore, Billerica, MA 1:500), anti-APP C-terminal antibody (Sigma-Aldrich 1.500), anti-Amyloid β (6E10, signet, Covance 1:1000) and mouse anti-βIII tubulin (1:1000, Promega Corporation) or mouse anti-β Actin (1:2000, Sigma-Aldrich) antibodies.

Techniques: