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. 2006 May;17(5):2465–2475. doi: 10.1091/mbc.E05-09-0826

Figure 3.

Figure 3.

Involvement of JRAB in the endocytic recycling of occludin in MTD-1A cells. (A) MTD-1A cells infected with Ad-EGFP (GFP), Ad-Myc-JRAB-F (JRAB-F), Ad-Myc-JRAB-N (JRAB-N), Ad-Myc-JRAB-C (JRAB-C), or Ad-EGFP-Rab13 Q67L (Rab13 Q67L) were cell surface biotinylated and incubated at 37°C for 15 min to allow for endocytosis of biotinylated occludin or TfR on the cell surface. After stripping the remaining biotin from the cell surface, cells were again incubated at 37°C for 15 min to allow recycling of biotinylated occludin or TfR back to the cell surface. After a second stripping of cell surface biotin, biotinylated occludin or TfR was isolated with avidin beads and quantitated by Western blot with anti-occludin or anti-TfR antibody. Recycled proteins represent the percentage of endocytosed proteins and expressed as the mean and SEM of three independent experiments. (B) MTD-1A cells infected with Ad-EGFP (GFP) or Ad-Myc-JRAB-N (JRAB-N) were cell surface biotinylated and incubated at 37°C for 15 min to allow for endocytosis of biotinylated occludin or TfR on the cell surface. After stripping the remaining biotin from the cell surface, biotinylated occludin or TfR was isolated and quantitated. Endocytosed proteins represent the percentage of endocytosed proteins and expressed as the mean and SEM of three independent experiments.