Modulation of 72-Kilodalton Type IV Collagenase: MATERIALS AND METHODS(3)
Ten micrograms of protein from conditioned media per lane was applied to a 10% SDS-polyacrylamide gel under nonreducing conditions. Protein electrotransference to Im-mobilon-P membranes (Millipore, Medford, MA) using a semi-dry system was performed according to Towbin et al.. Membrane was developed with monospecific polyclonal antibodies against 72-kDa type IV collagenase (MMP-2), generously donated by Dr. William Stetler-Ste-venson from National Institute of Cancer (NIH, Bethesda, MD). Primary antibodies were detected with the ABC method (Vector Labs., Burlingame, CA).
Total RNA from cells subjected to various ascorbic acid concentrations was extracted using Trizol (Gibco BRL). RNA was run in agarose gels and transferred to Immobilon-N membranes (Millipore). For collagen mRNA detection, poly-A was selected using oligo-dT cellulose according to previously described techniques. Membranes were hybridized with probes for mMp-2, type I collagen, and p-actin. The probes were labeled with the random primer method using [32P]CTP. Autoradiographies were quantitated with an EagleEye image analyzer (Stratagene, La Jolla, CA). The relative expression was normalized with p-actin mRNA. buy antibiotics online
WISH cells were grown in 25-cm2 culture flasks until 70% confluence; then fresh medium containing vitamin C was added as mentioned above. Nuclei from these cells were purified, and the newly transcribed RNA were labeled with [32P]UTP according to Davis et al.. DNA probes for MMP-2 and p-actin were transferred and cross-linked to Z-probe membranes (Bio-Rad) using a slot-blot apparatus. Labeled RNA were hybridized to immobilized probes and developed by autoradiography. The transcription rate was calculated by comparing the MMP-2 spots with the p-actin spots.