Cochlea from embryonic mice and mice up to 1 year old were removed and the pure membrane fraction loaded on 4-20% SDS gel, Following electrophoresis, transfer and blocking, the blot was incubated overnight at 4 degrees C with the GLUT10 primary antibody at a 1:500 dilution followed by incubation with secondary antibody at a 1:3,000 dilution for 1 h at room temperature. Protein bands were visualized on x-ray film with supersignal west femto chemiluminescent substrate. Lane 1. 10ug of protein from liver 2. 10ug of protein from cochlea (E18) 3. 10ug of protein from cochlea (P7) 4. 10ug of protein from cochlea (P10) 5. 10ug of protein from cochlea (P14) 6. 10ug of protein from cochlea (P17) 7. 10ug of protein from cochlea (P60) 8. 10ug of protein from cochlea (1 Year)
NCI-H1299 non-small cell lung tumor cells were transfected with the eukaryotic expression vectors pcDNA3.1/V5 (lanes 1,2), pCMV-GLUT10 (lanes 3,4), and pCMV-GLUT1 (lanes 5,6). GLUT10 was detetced using anti-GLUT10.
Dissected inner ear tissues from mice were immersed in 20% sucrose PBS solution (pH 7.4) overnight followed by being embedded in OCT overnight. The tissues were snap frozen in liquid nitrogen and 7um sections were cut by cryostat. Cochlear sections from the organ of Corti were dissected and rinsed by 0.1% Triton in PBS (pH 7.4, PBST) for 30 minutes. Sections were blocked with 5% goat serum in PBST at room temperature for 1 hour. The GLUT10 antibody was used at a 1:200 dilution to label the cochlear sections at 4 degrees C overnight. After washing, the sections were incubated with secondary antibodies (anti-rabbit Cy3, 1:400) at room temperature for 1 hour. Cy3-conjugated secondary antibodies were used to visualize the binding of the primary antibodies. The slides were mounted with fluoromont G and examined under a confocal microscope. Data from cochlear sections show strong immunoreactivity (stained red) is observed in the inner hair cells, outer hair cells and basilar membrane. In add
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