Enhanced chemiluminescence detection

Equal amounts of whole-cell lysates (15 μg) were separated on 10–12.5% polyacrylamid gels, transferred to polyvinylidene difluoride (PVDF) membranes, incubated with the indicated antibodies and visualized using horseradish peroxidase (HRP) conjugated secondary antibody (1:10 000, Jackson Immuno Research Laboratories, West Grove, PA, USA) followed by enhanced chemiluminescence detection (Biological Industries, Bet Haemek, Israel). Integrated optical densities of the bands were measured by Image reader Las3000, Multi-gauge v3.0 software. Optical densities were normalized to β tubulin or a general protein stain (ponceau).

Isolated splenocytes or islets were lysed in protein sample buffer (15% glycerol, 60 mM Tris-HCl pH 6.8, 3% SDS, 7.5% ß-mercaptoethanol). Protein lysates were separated on 10, 12.5 or 15% SDS-PAGE and transferred to a Protran nitrocellulose membrane (Whatman Ltd., Dassel, Germany) or to Immobilon PVDF membrane (Millipore Corporation, Billerica, MA). After blocking in 5% nonfat dry milk, the membranes were incubated with the appropriate antibodies, namely anti-tubulin (Sigma), anti-CD44 (eBioscience), anti-iNOS (Calbiochem, Merck, Darmstadt, Germany) or anti-cleaved caspase-3 (Cell Signaling, Denver, MA, USA) antibodies. All antibodies were diluted according to manufacturer instructions. Blots were exposed to secondary antibodies: anti-mouse IgG-HRP (Sigma) for tubulin detection, anti-rat-HRP (Nichirei Biosciences Inc.) for CD44 detection and anti-rabbit-HRP (Jackson ImmunoResearch) for iNOS and cleaved caspase-3 detections, followed by enhanced chemiluminescence detection (Biological Industries). Blots were developed using ECL (Molecular imager ChemiDoc XRS+ System, Bio-Rad, CA).

Western blotting was conducted as reported previously [19] (link). Briefly, proteins were quantified using the DC Protein Assay kit. After protein quantification and normalization, equivalent amounts of proteins were electrophoresed on 8 to 15% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Bedford, MA). After incubation with primary antibodies, the proteins were visualized by incubation with HRP-conjugated secondary antibodies followed by enhanced chemiluminescence detection (Biological Industries, BeitHaemek, Israel). The OPN polyclonal antibody (FL-314), RUNX2 polyclonal antibody (M-70), β-Actin polyclonal antibody (C-11) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The P21 Waf1/Cip1 polyclonal antibody (12D1), P27Kip1 polyclonal antibody (D69C12) were purchased from Cell Signaling Technology. The density analysis was performed for each WB band by using Quantity One 1-D analysis software (Bio-Rad Laboratories), and the average relative intensity value was shown along with the corresponding bands.