Targeted Disruption of Core 1 β1,3-galactosyltransferase (C1galt1) Induces Apical Endocytic Trafficking in Human Corneal Keratinocytes
Figure 2
Targeted disruption of C1galt1 enhances endocytosis of biotinylated cell surface protein in corneal keratinocytes.
(A) Cell surface protein was labeled with biotin at 4°C and then allowed to internalize for 15 or 25 min at 37°C. Crude postnuclear supernatants were ultracentrifuged using 5–25% Optiprep gradients and analyzed by agarose gel electrophoresis. Fraction 1 contains the lightest membranes; fraction 16 contains the densest membranes. The position of the plasma membrane and endocytic vesicles in the gradient was determined by western blot using antibodies to MUC16 and human TfR, respectively. Biotinylated protein was detected using streptavidin-peroxidase as described in Materials and Methods. (B) Quantitative evaluation of biotin accumulation in Optiprep density gradient. The graph shows the ratio of biotinylated protein in C1galt1 shRNA and scramble shRNA cells as determined by densitometry. Band intensity in each fraction was normalized to percent total biotin loaded in each gradient. Data for each condition are reported as the mean of three independent experiments.