Rabbit anti β actin antibody

Transduction of the TAT-14-3-3ε fusion protein was verified using Western blot analysis. Proteins were extracted from the cerebral hemisphere in cold RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% C₂₄H₄₀O₄·Na, 0.1% SDS, 2 mM Na₄P₂O₇, 25 mM β-glycerophosphate, 1 mM Na₃VO₄ and 1 mM PMSF) 4 h after intravenous injection of TAT-14-3-3ε or 14-3-3ε. Equal amounts of protein (40 μg) were diluted in 6×sample buffer, boiled and separated by 12% (wt/vol) SDS/PAGE, and then blotted onto PVDF membrane (Millipore, Billerica, MA, USA) using a wet transfer system (Bio-Rad, Hercules, CA, USA). The membrane was then immersed in blocking solution [5% milk in TBST (0.1% Tween 20 + TBS)] for 2 h at room temperature, followed incubated with a monoclonal rabbit anti-14-3-3ε antibody (1∶1000, Abcam, Cambridge, UK), rabbit anti-His-tag antibody (1∶1000, Abgent, CA, USA) or rabbit anti-β-actin antibody (1∶2000, Santa Cruz Biotechnology, CA, USA) overnight at 4°C. Subsequently, membranes were incubated with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1∶5000, Santa Cruz Biotechnology, CA, USA), followed by ECL detection (Pierce, Rockford, IL, USA). The density of each band was quantified using the Quantity One software.

Cells were seeded onto 10-cm dishes. total protein lysates were extracted in ice-cold radioimmunoprecipitation assay buffer and sonicated twice for 6 s each19 (link). Separation of Triton X-100-soluble and -insoluble fractions was performed according to a previous protocol. Protein concentrations were determined using a BCA protein assay kit. Protein lysates (100 mg) were separated on a 10% Bis–Tris polyacrylamide gel and subsequently transferred onto a polyvinylide fluoride membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 and probed with a rabbit anti-Runx2 (1:1000; Abcam ab102711), Col1(1:1000; Abcam ab34710), Osterix (1:1000; Abcam ab22552), and ALP (1:1000; Abcam ab95462) primary antibody followed by ALP-conjugated goat anti-rabbit IgG secondary antibody (1:10000; Santa Cruz sc-2004). Antibody detection was carried out using a BCIP/NBT kit (Zymed, Invitrogen) according to the manufacturer’s instructions. As a loading control, membranes were immunoblotted with a rabbit anti-β-actin antibody (1:1000; Santa Cruz sc-130657). Protein levels were quantified using ImageJ software (National Institutes of Health, MD, USA). After correcting for background, the integrated density of each protein band was measured using the same selection surface for each measurement19 (link).

First the samples were homogenized in 1xRIPA buffer [10 mmol/L Tris-HCl (pH 8.0), 140 mmol/L NaCl, 1 mmol/L EDTA (pH 8.0), 0.5 mmol/L EGTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate] containing protease inhibitors (Sigma-Aldrich). The homogenate was incubated on ice for 40 minutes and centrifuged at 15,000 g for 15 minutes. The protein concentration in supernatant was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). Samples at 50 μg/lane were separated on a 10% SDS-PAGE gel. The separated samples were transferred to nitrocellulose membranes and exposed to rabbit anti-HMGCR antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-β-actin antibody (1:5000; Santa Cruz Biotechnology) for overnight at 4 °C. After washing, the membranes were incubated with the secondary antibody, a horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Santa Cruz Biotechnology) for 1 hour at room temperature, and visualized with enhanced chemiluminescence reagents (Santa Cruz Biotechnology). The experiment was repeated three times.