Figure 6. Effect of Odin on EGFR recycling.

A, Western blot analysis of the whole cell lysates from lentivirus infected cells expressing a non-silencing control (shCont) or Odin-directed (shOdin) shRNA, or shOdin cells with stable ectopic expression of myc-Odin encoded by an shRNA-resistant Odin expression vector (srOdin). B, A schematic drawing showing from left to right the sequential experimental steps employed to measure EGFR degradation and internalization in shOdin, shCont and srOdin cells (monensin, mon; CHX, cyclohexamide, streptavidin affinity purification, SA-AP). C, Time-course of EGF stimulation of cells with basal (shCont), knocked down (shOdin) or rescued/over-expressed levels of Odin expression. Biotinylated EGFR was captured by using streptavidin (SA) beads, and then western blotted for EGFR. When cell surface biotin was removed prior to the SA adsorption step (+ cleavage), only internalized (i.e. protected from cleavage) EGFR are expected to be recovered, whereas in the absence of cleavage (- cleavage) both internalized and plasma membrane-localized EGFR would be captured. The cells were treated with monensin (Mon) to block receptor recycling. D, The effect of monensin on the amount of intracellular EGFR. Western blot analysis of biotin-labeled EGFR (captured by immobilized streptavidin) with indicated cleavage, EGF, and monensin treatment for 10 min. Results shown are representative of three independent experiments.