The preparation of cortical tissue extraction for immunoblotting was followed by the method described in our previous study63 (link). Protein extracts were subjected to 10% Bis-Tris gel (Invitrogen, Grand Island, NY, USA), incubated with 5% non-fat dry milk in TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature, and then followed by the primary antibodies with gently shaking overnight at 4 °C. The primary antibodies used were as follows: anti-EP (Cat# 36-3000, Invitrogen), anti-phos-p38 (Cat# 612288, BD), anti-total-p38 (Cat# 9212, Cell signaling), anti-human Aβ 1-17 clone 6E10 (Cat# 9320-02, Signet), anti-synaptophysin (Cat# MAB5258; Chemicon), anti-synaptojanin 1 antibody (AC1), (Cat# MA3-936; Thermo Fisher), anti-NMDAR2B (Cat# ab81271, Abcam), anti-BACE1 antibody (Cat# ab108394, Abcam), and β-actin (Cat# A5441; Sigma-Aldrich). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for quantification of intensity of the immunoreactive bands in the scanned blots.
Anti bace1 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-BACE1 antibody is a laboratory reagent that can be used to detect and measure the presence of the BACE1 protein in biological samples. BACE1 is an important enzyme involved in the production of amyloid-beta, which is a key component of the plaques found in the brains of individuals with Alzheimer's disease. This antibody can be a useful tool for researchers studying the role of BACE1 in various biological processes and disease states.
Automatically generated - may contain errors
Lab products found in correlation
Anti total p38, by Cell Signaling Technology (1 mentions)
Anti nmdar2b, by Abcam (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Anti actin antibody, by BD (1 mentions)
Quickchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti app antibody, by Abcam (1 mentions)
Anti adam9, by Cell Signaling Technology (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Anti β tubulin 3 neuronal antibody, by Merck Group (1 mentions)
4 protocols using anti bace1 antibody
1
Western Blot Analysis of Cortical Proteins
2
Generating Mutant CALHM1 Constructs
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Anti-myc antibody (71D01) was from Cell Signaling Technology, anti-actin antibody from BD Transduction Laboratories, anti-Aβ1–17 (6E10) antibody from Covance, and anti-BACE1 antibody from Abcam. Myc-tagged WT- and W114A-CALHM1 in pcDNA3.1 vectors, as well as WT-CALHM1 in the pBF oocyte expression vector, were described previously [9] (link), [13] (link). G330D- and R154H-CALHM1 were created using Quickchange II site-directed mutagenesis kit (Agilent Technologies) and confirmed by sequencing.
3
APP Processing Regulation Protocol
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Rabbit anti-APP antibodies to detection the C-terminal of APP were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). FBS was purchased from ATCC Company (Manassas, VA, USA). DMEM, penicillin/streptomycin, G418, and 0.25% trypsin-EDTA were purchased from GIBCO–BRL Company (Carlsbad, CA, USA). Zeocin were purchased from Invitrogen Company (Carlsbad, CA, USA). Rabbit anti-GAPDH, anti-rabbit horseradish peroxidase linked IgG, anti-ADAM9 antibodies and lysis buffer were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-BACE1 antibody, and anti-APP antibody to detection both mAPP and imAPP were obtained from Abcam Company (Cambridge, UK). Anti-ADAM10 antibody was obtained from Calbiochem Company (San Diego, CA, USA). Anti-TACE and anti-ADAM17 antibodies were obtained from Chemicon Company (Billerica, MA, USA). All other chemicals were of analytical grade obtained from Sigma-Aldrich Co.
4
Immunostaining of Cultured Neurons
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The culture medium was discarded, and the neurons were washed 3 times with PBS. The cultured neurons were perfused with 4% buffered paraformaldehyde (PFA) for 30 min, washed 3 times with PBS, and then incubated in blocking solution (0.1% Triton X-100 + 10% goat block serum + PBS) for 1 h. Following this step, the cultured neurons were incubated with the primary antibodies anti-β-Tubulin III (neuronal) antibody (Cat no. T8578; 1:5000; Sigma, Saint Louis, USA) or anti-BACE1 antibody (catalog #ab2077; 1:1000; Abcam) overnight at 4°C. The neurons were then washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. The immunofluorescence intensity of BACE1 was measured by image Plus 5.0 software.
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