Horseradish peroxidase conjugated goat anti mouse igg

U87-MG and U251 cells were collected and resuspended in ice-cold RIPA buffer. Cells were then lysed on ice for half an hour and centrifuged at 12,000 rpm for 30 min. Protein concentration was measured using BCA Protein Assay kit (Beyotime, Shanghai, China). Protein samples were diluted with loading buffer (Beyotime, Shanghai, China) and then heated at 100°C for 10 min. Equal amount of protein samples (20μg) were separated using a 10% sodium dodecyl sulfonate-polyacrylamide gel by electrophoresis. Protein content was then transferred onto PVDF membranes (Millipore, Billerica, MA, USA) and blocked with 5% not-fat milk at room temperature for 2 h. Blocked membranes were then incubated with anti-β-actin (1:2000; BioVision, Milpitas, CA, USA), anti-CDK6 (1:2000; Atlas Antibodies AB, Bromma, Sweden) or anti-ZEB1 (1:1000; Atlas Antibodies AB, Bromma, Sweden) at 4°C overnight. The following day, membranes were rinsed using 1×TBST and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5000, Cell Signaling Technology, Danvers, MA, USA) or anti-rabbit IgG (1:5000, Cell Signaling Technology, Danvers, MA, USA) at room temperature for 2 h. Protein bands were visualized using an enhanced chemiluminescence kit (ECL, Solarbo Life Sciences, Beijing, China) by exposure to X-ray films.

APPsw-transfected HeLa cells were washed with PBS and homogenized using lysis buffer (100 mM NaCl, 1% Triton X-100,1 mM sodium orthovanadate, protease inhibitors, 50 mM HEPES, pH 7.2). Centrifugation of cell lysates was carried out at 10,000× g for 10 min. The Bradford assay (Bio-Rad, Hercules, CA, USA) was used to measure protein concentration in the supernatant. SDS-PAGE (8% or 15%) was used to resolve proteins. The resolved proteins were transferred to a polyvinylidene fluoride membrane. Then, it was blocked using nonfat milk powder (5%) in Tris-buffered saline/Tween 20 (TBST) for 1 h at room temperature. The membrane was then incubated overnight at 4 °C with anti-sAPPα (Covance), anti-CTF (Sigma), anti-ADAM9 (Cell Signaling Technology, Danvers, MA, USA), anti-ADAM10 (Abcam), anti-ADAM17 (Abcam), anti-β-tubulin (Sigma), or β-actin (EnoGene, New York, USA). After washing membranes using TBST, horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Cell Signaling Technology) were incubated at room temperature for 1 h. Enhanced chemiluminescence was performed to visualize the peroxidase activity. Multi Gauge Software (Fujifilm, Tokyo, Japan) using a LAS-4000 system was used to quantify the detected signals.

Western blot analyses were performed to assess age- and DM-related changes in APP, Rab5, Rab7, Rab11, DHC, DIC, KHC, KLC, NEP, Cathepsin D heavy chain (CatD HC), and LC3 expression. Each microsome fraction prepared as described above was adjusted to 5 mg, and then analyzed using SDS-polyacrylamide gel electrophoresis using 14% (for LC3) or 12.5% (other proteins) acrylamide gels. Separated proteins were blotted onto polyvinylidense fluoride membranes (Immobilon P; Millipore). The membranes were blocked with 5% nonfat dried milk in 20 mM PBS (pH 7.0) and 0.1% Tween-20 for 1 h at room temperature, and then incubated overnight at 4°C in the following primary antibody solutions: (1) synaptophysin (1:20000); (2) APP (1:2000); (3) Rab5m (1:2000); (4) Rab7 (1:10000); (5) Rab11 (1:2000); (6) DHC (1:1000); (7) DIC (1:20,000); (8) KHC (1:5,000); (9) KLC (1:1000); (10) NEP (1:4,000); (11) CatD (1:2,000); or (12) LC3 (1:2,000). They were then incubated with either horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:10,000; Cell Signaling Technology) for 1 h at room temperature. Immunoreactive elements were visualized using enhanced chemiluminescence. To confirm reproducibility, immunoreactive bands obtained from the Western blots were quantified using commercially available software (Quantity One; PDI, Inc.).

Monoclonal antibodies against CBS and CSE were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-CD31 antibody was obtained from R&D Systems (Minneapolis, MN, USA). Prolong Gold antifade reagent with 4, 6-diamidino-2-phenylindole (DAPI), Alexa⁴⁸⁸ and Alexa⁵⁶⁸ conjugated goat anti-mouse immunoglobulin G (IgG) were obtained from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase-conjugated goat anti-mouse IgG was obtained from Cell Signaling (Beverly, MA, USA). ICI was from Tocris (Minneapolis, MN, USA). Bovine serum albumin (BSA) and all other chemicals, unless specified, were obtained from Sigma (St. Louis, MO, USA).

HAECs were lysed for 30 min on ice in lysis buffer (Bio-Rad, Richmond, CA) with 1 μg/mL protease inhibitors. Cell debris was removed by centrifugation (14,000 ×g, 4°C, 15 min), and supernatants were collected and stored at −80°C until use. Total protein samples (30 μg each) were boiled for 5 min, resolved by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis in Tris/glycine/SDS buffer (25 mM Tris, 250 mM glycine, and 0.1% SDS), and blotted onto transfer membranes (Bio-Rad, Richmond, CA) (100 V, 1.5 h, 4°C). After blocking for 2 h in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) containing 5% dry nonfat milk, the membranes were washed three times in TBST and probed for 18 h at 4°C with the anti-TLR2 monoclonal antibody TL2.1 (final concentration: 1 μg/mL; Abcam, Cambridge, MA) and anti-β actin monoclonal antibody (1 : 1000 dilution; Cell Signaling Technology, Beverly, MA) in TBST. After three washes in TBST, the membranes were incubated with horseradish-peroxidase-conjugated goat anti-mouse IgG (1 : 2000 dilution; Cell Signaling Technology, Beverly, MA) and then washed five times in TBST. Protein bands were detected using ECL reagents (GE Healthcare, Waukesha, WI) according to the manufacturer's instructions.

Dot blot analyses were performed to assess age- and DM-related changes in GAβ formation. Brain samples were adjusted to 1 mg, 2.5 mg, and 5 mg, and then applied onto nitrocellulose membranes and dried. The membranes were blocked with 5% nonfat dried milk in 20 mM PBS (pH 7.0) and 0.1% Tween-20 for 1 h at room temperature, and then incubated in the anti-GAβ antibody solution overnight at 4°C. They were then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:10000; Cell Signaling Technology) for 1 h at room temperature. Immunoreactive elements were visualized using enhanced chemiluminescence (Luminata Forte Western HRP Substrate, Millipore).