Pierce ecl western blot substrate

Rabbit polyclonal antibodies against the silkworm PPO (1: 5,000) (29 (link)), 30Kc19 (1: 3,000) (30 (link)), M. sexta βGRP-2 (1: 2,000) (31 (link)), Drosophila PPO1 (1:5,000) (27 (link)), and mouse polyclonal antibody against Drosophila PPO2 (1: 1,000) were used as the primary antibodies for Western blot analyses. Antibody binding was visualized by a color reaction catalyzed by alkaline phosphatase (AP) conjugated goat anti-rabbit/mouse IgG (1:10,000; Chemicon), or visualized by enhanced chemiluminescence catalyzed by horseradish peroxidase (HRP) using Pierce ECL Western blot substrate (Thermo).

After the mice were deeply anesthetized and decapitated; both brain hemispheres were collected and stored in −80°C. The frozen hippocampus and cortex of mice brain were homogenized in prechilled radioimmunoprecipitation assay (RIPA) buffer. The protein concentration of the samples was determined with the BCA kit (Pierce, MA, USA). The same amounts (20–30 μg) of brain protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 8, 10, or 12% gels and then transferred on to a polyvinylidene difluoride membranes (Roche, Switzerland). The membranes were incubated overnight at 4°C with different primary antibodies, which are list in Table 1. We then incubated the membranes with secondary antibody (1:3,000, Cell Signaling Technology, Danvers, MA, USA) conjugated to horseradish peroxidase (HRP) for 1 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and b-tubulin levels were analyzed as a loading control. The immunocomplexes were visualized using the Pierce ECL Western blot substrate (Thermo Scientific, Waltham, MA, USA). We visualized the protein bands with automatic chemiluminescence apparatus (BIO-RAD, USA). The specific molecular band intensity was determined using ImageJ.

R1 cells were grown in 6-well plates seeded with CD1 MEFs as feeders, and treated by lentiviruses as described in the Apoptosis and cell cycle assays section. Total cellular proteins were then extracted using RIPA buffer (Thermo Fisher Scientific) with 1× proteinase and phosphatase inhibitors (Thermo Fisher Scientific). Proteins were quantified with a BCA-Quantification kit (Thermo Fisher Scientific), and subjected to 10% SDS-PAGE gel electrophoresis using BioRad mini-gel system and subsequently transferred to PVDF membranes.
The blotted membranes were then blocked with 5% non-fat dry milk in TBS-T and incubated with primary antibodies at 4°C overnight. The antibodies used were as follows: anti-GAPDH (1/2500, Abcam). Antibodies against p53, Mdm2, pMdm2, and pGSK3β (1/1000 for all) were from Cell Signaling. All Akt isoforms, phospho-Akt Ser473, and pan-Akt were detected using Akt isoform antibody sampler kit from Cell Signaling. Membranes were then washed and blotted with HRP conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Chemiluminescence was detected either using Pierce ECL Western-Blot Substrate (Thermo Fisher Scientific) and X-ray film exposure, and quantified using the ImageJ software (Schneider et al., 2012 (link)), or detected and quantified using BioRad Gel Doc XR System (Hercules, CA, USA).

HFLS cells were lysed with RIPA lysate (Beyotime). Total proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were incubated with 5% skimmed milk for 1 h, and incubated overnight at 4℃ with primary antibodies against interleukin‐1β (IL‐1β) (1:1000, ab216995; Abcam), interleukin‐6 (IL‐6) (1:1000, ab233706; Abcam), tumour necrosis factor‐α (TNF‐α) (1:1000, ab215188; Abcam), and GAPDH (1:1000, ab8245; Abcam). After washing, the membranes were incubated with a secondary antibody (1:5,000; Biotech) at room temperature for 4 h. The signals were developed using Pierce ECL Western Blot Substrate (Thermo Fisher Scientific), imaged, and analyzed with GAPDH as the control using ImageJ software.

Western blotting was performed using Hoefer Scientific Instrument apparatus and BioRad Western Blot electrophoresis system. 20-30 μg of protein was resolved using sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Proteins were transferred onto a nitrocellulose membrane (Millipore-Immobilon), which was then blocked in 5% milk-TBST for 1 hour at room temperature (RT) and incubated overnight at 4°C with the relevant primary antibody (see Key Resources Table). The following day, 3 washes in TBS-T were performed before incubation with the appropriate HRP-conjugated secondary antibody (anti-mouse, GE Healthcare NA931; antirabbit, GE Healthcare NA934; anti-chicken, Sigma-Aldrich AP194P) for 1 hour at RT at 1/5000. After 3 additional washes in TBS-T, proteins of interest were detected with Pierce- ECL western blot substrate (Thermo Scientific) or Luminata Crescendo Western HRP substrate (EMD-Millipore) and images acquired on the Imagequant LAS 4000.

Experiments using primary cells were conducted using the following numbers of cells: 4 × 10⁸ human platelets per immunoprecipitation and 1 × 10⁷ per lane of whole cell lysate; 1.6 × 10⁸ mouse platelets per immunoprecipitation and 4 × 10⁶ per whole cell lysate; and 2.2 × 10⁶ HUVECs per immunoprecipitation and 5.5 × 10⁴ per whole cell lysate. Whole cell protein lysates and co-immunoprecipitation experiments were performed as previously described (8 (link)). Briefly, cells were lysed in 1% digitonin lysis buffer (10 mm Tris, pH 7.4, 150 mm NaCl, 0.02% NaN₃). Proteins were immunoprecipitated with primary antibody (as indicated in text) bound to protein G-Sepharose beads for 90 min and washed in 0.1% digitonin lysis buffer. Standard protocols were used for Western blotting and SDS-PAGE. Primary antibodies were used as indicated in the text with corresponding horseradish peroxide (Pierce) or IRDye® 680RD or 800CW (LI-COR Biosciences)-conjugated secondary antibodies. Membranes were visualized using Pierce ECL Western blot substrate (Thermo Scientific) and exposure to film or using an Odyssey Infrared Imager (LI-COR Biosciences). All quantitation was performed using an Odyssey Infrared Imager (LI-COR Biosciences); background signal was removed, and individual band intensities were compared.