Anti osteopontin opn

The tumor samples were collected from each patient during surgical treatment. Samples were homogenized and lysed. Extracts were resolved on SDS-polyacrylamide gels followed by transfer to nitrocellulose membranes. Proteins were resolved by electrophoresis on 8–12% sodium dodecyl sulfate–polyacrylamide gels and transferred by electroblotting to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk and incubated overnight at 4 °C with the anti-NOTCH3 (Santa Cruz, 1:1000), anti-Osteopontin (OPN, Santa Cruz, 1:1000), anti-NF-kB (Santa Cruz, 1:1000) and anti-GAPDH (Santa Cruz, 1:2000) antibodies. The anti-human IgG was used as negative control. Immunolabeling was detected using the enhanced chemiluminescent Reagent (Amersham Biosciences, Buckinghamshire, UK).

The intervertebral discs were collected during surgery from patients during ACF treatment. Samples were homogenized and lysed. Extracts were resolved on SDS-polyacrylamide gels followed by transfer to nitrocellulose membranes. Proteins were resolved by electrophoresis on 8–12% sodium dodecyl sulfate–polyacrylamide gels and transferred by electroblotting to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk and incubated overnight at 4°C with the anti-HIF-1α (Novus Biological, 1;1000), anti-vascular endothelial growth factor (anti-VEGF) (Santa Cruz, 1∶1000), anti-VEGF receptor (anti-VEGFR) (Santa Cruz, 1∶1000), anti-NF-kB (Santa Cruz, 1∶1000), anti-interleukin 1 (anti-IL1) (Santa Cruz, 1∶1000), anti-interleukin6 (anti-IL6) (Santa Cruz, 1∶1000), anti-Osteopontin (OPN) (Santa Cruz, 1∶1000), anti-Osteoprotegerin (OPG) and anti-GAPDH (Santa Cruz, 1∶2000), antibodies. Immunolabeling was detected using the enhanced chemiluminescence Reagent (Amersham Biosciences).