Biomax xar film

Osteolytic bone lesions in mice were assessed by X-ray radiography. Anesthetized mice were placed on individually wrapped films (BIOMAX XAR Film, Cat#: F5763-50EA, Sigma-Aldrich) and exposed to X-ray radiography at 35 kV for 15 seconds using a MX-20 Faxitron instrument. Films were developed using a Konica SRX-101A processor. Changes in bone remodeling and osteolytic lesions (radiolucent lesions) in the hind limbs of mice were identified and quantified using the Adobe Photoshop software (Adobe Systems Inc.)

The supernatant in NuPAGE buffer was incubated at 70°C for 5 min at 1100 rpm shaking. The tube was placed on a magnetic stand to remove the beads, and the supernatant was loaded on the SDS-PAGE gel. The SDS-PAGE was performed using 4-12% NuPAGE Bis-Tris gel (Life Technologies, NP0321BOX) following the manufacturer’s protocol with MOPS running buffer (Life Technologies, NP0001), NuPAGE Antioxidant (Life Technologies, NP0005) was added to the buffer in the upper chamber, and PAGE was performed for 45 min at 180 V. 5 μl of PAGE ruler plus (Thermo Scientific, SM1811) was used as protein size marker.
After the run, the dye front of the gel was removed and discarded since this part contained free radioactive ATP. The protein-RNA complexes from the gel were transferred to a Protran BA85 Nitrocellulose Membrane (Whatman) using the Novex wet transfer apparatus following the manufacturer’s protocol (Life Technologies; transfer for 1 h at 30 V using NuPAGE transfer buffer (Life Technologies, NP0006-1, supplemented with 10% methanol). The membrane was wrapped with saran wrap and exposed to a Carestream Kodak BioMax XAR Film (SIGMA, Z358487) at −80°C for 1 hour and overnight.