PA1-757

antibody from Invitrogen Antibodies

Targeting: BACE1 BACE

Provider product page for PA1-757

Western blot

Immunocytochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [35]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [3]
Other assay [39]

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Product number: PA1-757 - Provider product page
Provider: Invitrogen Antibodies
Product name: BACE1 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-757 detects transfected beta-secretase 1.
Concentration: 1 mg/mL

BACE1 protein structure - PA1-757 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of BACE1 (green) showing staining in the Golgi apparatus of HeLa cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a BACE1 polyclonal antibody (Product # PA1-757) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

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Main image
Experimental details: Immunofluorescent analysis of BACE1 (green) showing staining in the Golgi apparatus of Neuro-2a cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a BACE1 polyclonal antibody (Product # PA1-757) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of BACE1 (green) showing staining in the Golgi apparatus of SH-SY5Y cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a BACE1 polyclonal antibody (Product # PA1-757) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

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Supportive validation

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Experimental details: Fig. 6. Downregulation of calsyntenin-1 by RNAi results in enhanced APP processing and increased secretion of Abeta. Cortical neurons were infected with rAAVs expressing a hairpin targeting calsyntenin-1 (shCst1) or a nonsense hairpin (shNS), together with eGFP. ( A ) Western blot of cell lysates. The signal indicated equal expression of the recombinant proteins. ( B ) Quantification of Western blots. Values indicate mean+-s.e.m. (*, P

Supportive validation

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Experimental details: Figure 2 Combinations of pro-inflammatory cytokines elevate endogenous BACE1 levels in mouse primary astrocyte cultures . (A, B) Cell lysates of cytokine-stimulated mouse primary astrocytes analyzed in Fig. 1A-C were analyzed for BACE1 by immunoblot. (A) BACE1 immunoblot images. The mature BACE1 band at 70 kDa is indicated by the arrowhead. Pro-inflammatory agents (alone and combinations) and stimulation times are indicated. Cell lysate from un-stimulated BACE1 -/- primary astrocytes was used as a negative control, while cell lysate from a stable BACE1-overexpressing HEK-293 cell line was used as a positive control. GFAP and beta-actin immunosignals were loading controls as in Fig 1A-C. (B) Histograms represent quantifications of BACE1 immunoblot signals in (A) expressed as percent of vehicle control. Note that cytokine combinations including TNF-alpha and IFN-gamma were generally more potent at increasing endogenous BACE1 levels in astrocytes over time in culture, raising BACE1 levels to ~600% of control. (C) mRNAs prepared from cytokine-stimulated primary astrocytes in Fig 1D were analyzed for endogenous BACE1 mRNA levels by TaqMan quantitative RT-PCR. Histograms represent quantifications of BACE1 mRNA levels expressed as percent of vehicle control. Note that astrocytic BACE1 mRNA levels were significantly reduced by TNF-alpha+IFN-gamma stimulation (C), even though BACE1 protein levels were increased several fold in similarly treated astrocytes (B). Statistical analysis was

Supportive validation

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Experimental details: Figure 7 Oligomeric and fibrillar Abeta42 increase levels of endogenous BACE1 in mouse primary astrocyte cultures . Lysates of C57BL/6J mouse primary astrocyte cultures treated with 10 muM oligomeric or fibrillar Abeta42 from Fig. 6 were prepared for BACE1 immunoblot (A, B) or mRNA quantification by TaqMan RT-PCR (C). Treatment times are indicated. Olig., Abeta42 oligomer treatment; Fibr., Abeta42 fibril treatment; OC, oligomer vehicle control; FC, fibril vehicle control. Cell lysate from un-treated BACE1 -/- primary astrocytes was used as a negative control, while cell lysate from a stable BACE1-overexpressing HEK-293 cell line was used as a positive control. GFAP immunosignals were loading controls as in Fig. 6A. Abeta42 oligomers and fibrils elevated BACE1 protein levels at 48 h and 96 h of treatment (A, B). BACE1 levels peaked at nearly 300% of control at 96 h that correlated with a rise in BACE1 mRNA level (C). Unlike APP, levels of both BACE1 protein and mRNA did not return to normal but remained elevated at 96 h. Asterisks indicate significant differences as compared to vehicle controls (*: p < 0.05; **: p < 0.01; n = 3). Error bars, SEM.

Supportive validation

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Experimental details: Figure 3 Minocycline inhibits beta-site APP cleaving enzyme 1 in young, pre-plaque Tg mice . (A) BACE-1 activity was quantified from hippocampal homogenates using a well-characterized fluorometric assay. Fluorescent units (FU) data were normalized on Non Tg Placebo values. Note the significant up-regulation of BACE-1 activity in Tg Placebo compared to Non Tg Placebo (*** P < 0.001), which was corrected following minocycline treatment (* P < 0.05 compared to Tg Placebo). (B) Specificity of the BACE-1 fluorometric assay: note how the fluorescent units detected from brain homogenates (of a Non Tg Placebo animal) are seven times higher than the background (bg). The signal was completely abolished by co-incubation with a specific BACE inhibitor (BACEi), and was in the range of activity of a recombinant human BACE protein (rhBACE, 2.5 mug). All the data were analyzed with one-way ANOVA followed by Tukey post-hoc test. (C) Specificity of the polyclonal BACE-1 antibody used for western blotting: the specific band (close to 75 kDa) appeared in brain extracts from post natal day 7 (pnd 7) and from adult cortex (ctx), while it was hardly detectable in cerebellum (cereb) sample and was not detectable in a glial preparation from pups. (D) Representative western blots of BACE-1, NFkB and IkB from the same hippocampal samples used for the enzymatic assay (ns = non-specific band recognized by the polyclonal antibody directed against NFkB). (E) Quantification data of BACE-1, NFkB and IkB from

Supportive validation

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Experimental details: Figure 3 BACE1 protein level and activity are down-regulated by Tyro3 overexpression in 293APPswe cells. (A, B) Western blot analysis of BACE1 showing that the protein levels of BACE1 in extracts from Tyro3-CFP transfected cells have lower fluorescent intensity than GFP controls. (C, D, E) Increase in C99 (betaCTF) protein level and decrease in C83 (alphaCTF) protein level in extracts from Tyro3-CFP transfected cells were detected by western blot analysis compared to that of GFP control. * P

Supportive validation

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Experimental details: Figure 4 Gas6 inhibits the decrease in BACE1 protein level induced by Tyro3 overexpression in 293APPswe cells. (A, B) Western blot statistical analysis showed that BACE1 levels were significantly lower in Tyro 3-CFP transfected cells than in GFP controls. Gas6 treatment was found to reverse this effect. * P

Supportive validation

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Experimental details: Fig. 2. Calsyntenin-1/APP/BACE1 transport packages are found along axons and in growth cones. Immunofluorescence staining of dissociated hippocampal neurons. Black-and-white masks (a4-d4) were generated to emphasize the colocalization in axons ( A ) and growth cones ( B ). Inserts (a4-d4) show stainings of the axonal marker Tau-1. Note that BACE1 (a1-d1) colocalizes with calsyntenin-1 (a2,a3), APP (b2,b3), and Stx13 (c2,c3), but not with Rip11 (d2,d3). ( C ) Schematic representation of the C- and P-region of the growth cone. ( D ) Fractions of BACE1 puncta that are colocalized with calsyntenin-1, APP, syntaxin13, and Rip11 puncta. Note that BACE1 colocalizes with calsyntenin-1, APP, and syntaxin13 along axons and in the C-domain of growth cones. ( E ) Fractions of calsyntenin-1, APP, syntaxin13, and Rip11 puncta that are colocalized with BACE1 puncta. Note that the colocalization with BACE1 is confined to axons and the C-domain of growth cones. Values represent mean+-s.e.m. (*, P

Supportive validation

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Experimental details: Fig. 7 Reduced APP proteolytic processing in APP/PS1/FTnKO mice. A , B Immunoblots and densitometric analysis of full-length APP (FL-APP), and APP cleavage products, carboxyl-terminal fragments (CTFalpha and CTFbeta), amino-terminal fragments of APP (sAPPalpha and sAPPbeta), and beta-secretase 1 (BACE1). Protein levels were normalized by the amount of tubulin with the levels in the APP/PS1 control group set as 100%. C , D Immunoblots and densitometric analysis of proteins involved in Abeta clearance pathways including APOE, LRP1, IDE, and neprilysin. Protein levels were normalized by the amount of tubulin or GAPDH, and the levels in the control group set as 100%. n = 8/genotype; Student's t-test, 2-tailed; # p = 0.08; * p < 0.05

Supportive validation

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Experimental details: Figure 8 Oligomeric and fibrillar Abeta42 increase levels of APPsbetasw in Tg2576 transgenic mouse primary astrocytes . Tg2576 or non-transgenic (non-Tg) littermate mouse primary astrocyte cultures were treated with 10 muM oligomeric or fibrillar Abeta42. Following treatment, cells were harvested for APP and APPsbetasw immunoblots. Treatment times are indicated. Olig., Abeta42 oligomer treatment; Fibr., Abeta42 fibril treatment; OC, oligomer vehicle control; FC, fibril vehicle control. The numbers in parentheses indicate primary astrocytes cultured from different Tg2576 mouse pups. Immunosignals identified by the anti-APP antibody 22C11 clearly demonstrated that Tg2576 astrocytes overexpress transgene-derived APPsw (A-C, upper row). Note that Tg2576 APPsw migrates faster than non-Tg astrocytic APP on SDS-PAGE because the Tg2576 transgene expresses the 695 amino acid form of APP, while endogenous APP expressed by astrocytes is the 751 amino acid form. Abeta42 oligomers and fibrils caused both transgenic APPsw and endogenous astrocytic APP to become elevated at 24 and 48 h (A, B). The APPsw increase largely returned to control levels by 72 h for Tg2576 astrocytes (C). Most importantly, oligomeric and fibrillar Abeta42 treatment dramatically induced beta-secretase processing of APPsw to generate robust levels of APPsbetasw in Tg2576 astrocytes (A-C, center row), as indicated by immunosignals produced by an antibody that recognizes the BACE1-cleaved C-terminal neo-epitope of A

Supportive validation

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Experimental details: Fig. 1. Calsyntenin-1/APP vesicles contain BACE1, but no ADAM10. ( A ) Schematic representation of vesicle immunoisolation. Vesicle immunoisolations were performed with antibodies against cytosolic epitopes of vesicle-associated proteins and Dynabeads coated with protein A. Captured vesicles were isolated and washed. Vesicular proteins were eluted by lysis of the captured vesicles with a mild detergent. Note that the vesicular component bearing the antigen targeted for immunoisolation is not or only partially found in the eluate, because of its strong binding to the antibody/protein A/bead complex. Its complete elution required harsher conditions, e.g. boiling of the beads in SDS. Therefore, the antigen used for vesicle immunoisolation is not shown on the Western blots of the (mild) eluates. Examples of antigen elutions under harsh condition from the beads are shown in the panels labeled beads. ( B ) Specificity tests of anti-APP, anti-ADAM10, and anti-BACE1 antibodies, performed by the addition of ~25 ug of recombinant antigen (GST-APPcyto, GST-ADAM10cyto, BACE1[485-501]). Saturation with recombinant antigen prevented vesicle capture. ( C ) Calsyntenin-1 organelles contained BACE1, but no ADAM10 (black box). ( D ) APP and syntaxin13 immunoisolates contain a low level of active ADAM10 and abundant BACE1. Neither BACE1 nor ADAM10 were found in Rab11 immunoisolates. ( E ) Rab5 immunoisolates contain a small amount of active ADAM10 (arrowhead) and abundant BACE1 (green box). ( F

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. The beta-secretase BACE1 interacts with gamma-secretase at endogenous levels in WT mouse brain microsomal lysate. (A) 1% CHAPSO-solubilized lysates of WT mouse brain microsomes were immunoprecipitated for BACE1 or ITGalpha1, ITGalpha2, and ITGbeta1 as controls. IPs were blotted to probe for coIP of the gamma-components NCT and PS1-NTF. (B) 1% CHAPSO-solubilized lysates of WT mouse brain microsomes were loaded onto BN-PAGE followed by second-dimension SDS-PAGE and blotted with antibodies to the indicated proteins. *, nonspecific signal; m and im, mature and immature forms.

Supportive validation

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Experimental details: Figure 2. BACE1 exists with gamma-secretase in an HMW complex isolated from mouse brain by FPLC and has proteolytic activity. (A) 1% CHAPSO-solubilized WT mouse brain microsomes were fractionated on a Superose 6 Increase SEC column by FPLC and blotted with antibodies to the indicated proteins. (B) FPLC fractions were pooled into HMW (6-9) or LMW (21-24) fractions and immunoprecipitated for BACE1 and then blotted with antibodies to the indicated proteins. Note that the amount of BACE1 that was immunoprecipitated varies and may not necessarily reflect the amount in the input. (C) FPLC fractions were blotted with certain antibodies to APP (top two panels, C7; bottom panel, 1G6). (D) FPLC fractions were incubated with a fluorogenic BACE1 peptide substrate. After incubation for 2 h at 37degC, fluorescence was read as BACE1 activity ( n = 3; error bars are SD). (E) 0.25% CHAPSO-solubilized WT mouse brain microsomes were immunoprecipitated for gamma-secretase (anti-NCT) in the absence or presence of two different beta-secretase inhibitors (AZD3293 and inhibitor IV, each at 10 uM), or else anti-TfR as a control. beta-Secretase activity assays were performed on the eluates extracted from the IP resin. n = 5 for the NCT IP, n = 2 for the TfR IP; error bars are SD. Unpaired Student's t test: **, P < 0.01; ***, P < 0.001. (F) Pooled HMW and LMW FPLC fractions from WT mouse brains were immunoprecipitated for gamma-secretase (anti-NCT). beta-Secretase activity assays were performed on the