Ecl plus reagent

Cells were lysed with raidoimmunoprecipitation assay (RIPA) reagent (Beyotime, ShangHai, China) in the presence of protease inhibitors (Sigma, USA) and total protein quantified with the bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). A total of 20 μg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and electro-transferred onto a polyvinylidene fluoride (PVDF) membrane (BioRad, Hercules, CA, USA). Expression of cleaved caspase 8 was detected using mouse anti-human caspase 8 monoclonal antibody (Cell Signaling Technology, Beverly, MA) and HRP-conjugated sheep anti-mouse antibody (Amersham Bioscience). Expression of cleaved PARP was detected using rabbit anti-human PARP polyclonal antibody (Cell Signaling Technology) and horseradish peroxidase (HRP)-conjugated sheep anti-rabbit antibody (Amersham Bioscience). Expression of GAPDH was detected using rabbit anti-human GAPDH polyclonal antibody (Amersham Bioscience) and HRP-conjugated sheep anti-rabbit antibody (Amersham Bioscience). Protein expression was visualized using ECL Plus reagent (Amersham Bioscience) and a Chemilumino analyzer GelDoc XR system (Biorad, USA).

For western blotting, rBMSCs were seeded in 6-well plates at a density of 1×10⁵ cells/well and cultured in DMEM with 2 μM quercetin for 0, 30, 60, 90, 120 and 150 min. The cells were then collected and lysed with a protein extraction reagent containing protease inhibitor cocktail, phosphatase inhibitor cocktail and phenylmethanesulfonyl fluoride (PMSF) (Kangchen, China). Equal amounts of protein samples were separated on duplicate 8% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% skim milk and incubated with the appropriate primary antibodies, including rabbit anti-rat ERK, p38, JNK, phosphorylated-ERK (p-ERK), phosphorylated-p38 (p-p38) and phosphorylated-JNK (p-JNK) (dilution, 1:1000; CST, USA), and mouse anti-rat actin (dilution, 1:5000; Sigma, USA). The membranes were then washed three times with PBS containing 0.1% tween-20 detergent and incubated for 2 hours with horseradish peroxidase (HRP)-conjugated goat anti-rabbit and rabbit anti-mouse secondary antibodies (Beyotime, China). Finally, the protein bands were visualized using ECL plus reagent (Amersham Pharmacia Biotech, USA) on a UVItec ALLIANCE 4.7 gel-imaging system. All experiments were performed in triplicate.

Protein concentrations were
determined using BCA assay (Sigma, U.K.). SDS-PAGE and western blotting
were performed under standard conditions using 50 μg of the
protein lysate per lane; proteins were separated on a 10–20%
gel and transferred to a PVDF membrane (Amersham, U.K.) using a wet
transfer blotter. The PVDF membrane was blocked with 5% dry milk in
TBS-T (Tris–borate–saline solution containing 0.1% Tween
20). Primary and secondary antibody incubations were performed using
TBS-T containing 0.5% dry milk or 5% BSA at RT for 1 h or at 4 °C
overnight. Membranes were developed using an ECL plus reagent (Amersham,
U.K.) for 5 min and were photographed using Kodak X-ray films in a
dark room, as performed previously.^{17 (link),19 (link)}

Immunoblot analysis and cell sample harvesting were performed as presented previously [40 (link)]. Briefly, ES cells and/or EBs were washed twice with PBS (pH 7.2) and lysed in sodium dodecyl sulphate (SDS) lysis buffer (50 mM Tris-HCl, pH 7.5; 1% SDS; 10% glycerol). Protein concentrations were determined using the DC protein assay kit (Bio-Rad, USA). Lysates were supplemented with bromophenol blue (0.01%) and β-mercaptoethanol (1%) and incubated for 5 min at 95°C. Equal amounts of total protein (10 μg) were subjected to 8 or 10% SDS-PAGE. After being electrotransferred onto polyvinylidene difluoride membrane (Immobilon-P, Millipore, USA), proteins were immunodetected using appropriate primary and secondary antibodies and visualized by ECL Plus reagent (Amersham Pharmacia Biotech, USA) according to manufacturer's instructions. We used the following primary antibodies: rabbit polyclonal antibodies against p38alpha kinase, Oct4 (Santa Cruz Biotechnology, USA), phospho-p38 kinase, and GAPDH (Cell Signaling Technology, USA). After immunodetection, each membrane was stained by Amido black to confirm equal protein loading. The total level of β-actin was detected as loading control.

Ten to 50 micrograms of protein were electrophoresed on 8–12% v/v polyacrylamide SDS-PAGE gels. Proteins were electrotransfered onto PVDF membranes. The membranes were subsequently blocked and incubated at room temperature with antibodies (1∶500 #sc-13067 Santa Cruz PGC-1 (H-300), 1∶2000 #ab110304 Abcam Sirt-1, 1∶2000 #sc-99 Santa Cruz p53 (Pab 240), 1∶2000 #06–758 Upstate ac.p53 (Lys 373,382), 1∶1000 #sc-33771 Santa Cruz NRF-1 (H-300), 1∶500 #sc-30963 Santa Cruz mtTFA (E-16)/TFAM/, 1∶1000 #sc-492 Santa Cruz Blc-2 (N-19), 1∶1000 #sc-525 Santa Cruz Bax (P-19), 1∶1000 #sc-27907 Santa Cruz Odf-1 (G-17), 1∶2000 #sc-377305 Santa Cruz LDH-C (H-65), 1: 5000#sc1616 Santa Cruz Actin (I-19). After incubation with primary antibodies, membranes were washed in TBS-Tween-20 and incubated with HRP-conjugated secondary antibodies. After incubation with the secondary antibody, membranes were repeatedly washed. Membranes were incubated with an ECL Plus reagent (RPN 2132, Amersham) and protein bands were visualized on X-ray films. The bands were quantified by ImageJ software (http://imagej.nih.gov/ij/) and normalized to beta-actin, which served as an internal control.

The protein samples were heated at 95°C for 10 min with the sample buffer (250 mM Tris-HCl, 4% sodium dodecyl sulfate, 2% β-mercaptoethanol, 10% glycerol and 0.003% bromophenol blue) and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein samples were then transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA), which were blocked with 5% skimmed dry milk in Tris-buffered saline (TBS) with 0.1 % Tween 20 (TBS-T) for 1 h. The membranes were then incubated overnight at 4°C with primary antibody diluted in 0.3% bovine serum albumin (BSA)-TBS-T. The membranes were incubated with the primary antibodies (CDK6, 1:1,000 and β-actin, 1:400; Abcam, Cambridge, UK) at 4°C for 12 h, and then with the horseradish peroxidase-linked secondary antibodies (1:1,000, goat anti-mouse monoclonal CD151 and goat anti-rabbit monoclonal β-actin; Abcam) at 37° for 1 h. The membranes were incubated with ECL Plus reagent (Amersham Biosciences, Uppsala, Sweden) and scanned using the Storm imaging system (Amersham Biosciences). Immunoreactive products were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA) by determining the optical density of the protein bands.