Chemiluminescence detection system

Protein samples were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes [9 (link)]. Membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) with 0.1% tween (TBST) and incubated individually with the following antibodies: Antibodies against HMGB1, RAGE, phospho (p)-NFκB p65, NFκB p65, cyclooxygenase (COX)2, tumor necrosis factor (TNF)α, TNF receptor (TNFR)1, IL-1β, IL-6, TLR4, TLR2, p- (ERK)1/2, ERK1/2, IFNγ, and cluster of differentiation (CD)68. All the antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) or Cell Signaling Technology, Inc. (Danvers, MA, USA) and used at a dilution of 1:1000. After washing for three times with TBST, the membranes were incubated with appropriate horseradish-peroxidase (HRP) conjugated secondary antibodies for 1 h at room temperature. Further, the membranes were washed three times with TBST and then developed using a chemiluminescence detection system (Amersham Biosciences, Buckinghamshire, UK). The blots were scanned and the signals were quantified by densitometric analysis using Image Studio Digits ver. 4 (Superior Street, Lincoln, Nebraska, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as control protein.

The pancreatic sample was lysed using RIPA lysis buffer on ice and centrifuged at 10,001× g (4 °C, 10min). The protein concentration of the supernatants was determined by the BCA Kit. For western blot analysis, the protein was separated by sodium dodecyl sulfate polypropylene gel electrophoresis (SDS-PAGE) and then electrotransferred to polyvinylidene difluoride (PVDF) membranes. All membranes were incubated with 50 g/L non-fat milk powder in triethanolamine buffer for 1 h at room temperature and then with primary antibodies against FasL, Cyt-c, caspase-3 (diluted 1:1000), and β-actin (diluted 1:1500) at 4 °C overnight. The following day membranes were washed with tris-buffered saline containing Tween-20 (TBST), then incubated with peroxidase-conjugated anti-rabbit secondary antibody for 1 h at room temperature. After washing with TBST three times, ECL reagent was added, and images were captured using a chemiluminescence detection system (Amersham Pharmacia, Piscataway, NJ, USA).

MG63 osteoblasts were harvested and rinsed with cold PBS. Cells were lysed and homogenized. Then the lyastes were spined at 14000 rpm for 10 min at 4°C. Protein concentration of total protein extracted was determined by the BCA Protein Assay (Pierce Chemicals Co., Rockford, IL, USA). The protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore, MA, USA) using a semidry transfer apparatus. Membrane was probed with anti-MMP1, anti-MMP2, anti-RANKL, anti-OPG (Bioworld, GA, USA), anticollagen I, anti-OCN antibody (Abcam, MA, USA), and anti-GAPDH inner control antibody (Sigma, MO, USA). And then the blots were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The immune complexes were visualized with a chemiluminescence detection system (Amersham Bioscience, NJ, USA).

The cells were lysed using RIAP lysis buffer and the concentration was measured by BCA method. Equal amount of protein was resolved using 12% SDS-polyacrylamide gel. The proteins were transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk and incubated with primary antibodies and HRP-conjugated goat anti-rabbit IgG. The proteins were tested using the chemiluminescence detection system (Amersham, Berkshire, UK). Finally, the bands were analyzed using ImageJ software.