MIB1 quantitatively reduces RYK surface expression in the absence of protein degradation. (A, B, D, and E) Histograms of individual cell fluorescence intensities depicting the distribution of surface labeling of RYK in nonpermeabilized HEK293T cells treated for 2 h with chloroquine and MG132, labeled, and then analyzed by flow cytometry. (A) Histogram shows the background of anti-HA immunoreactivity in vector only as compared with glue-RYK–transfected cells. Background intensity was used to determine the RYK-positive gate in each experiment individually. (B) Histogram of surface RYK expression on cells transfected with glue-RYK and vector only or flag-MIB1 or flag-MIB1 mutants. (C) Graph represents the number of cells in the RYK-positive gate shown in B. Data demonstrate a significant decrease in surface RYK expression when cotransfected with MIB1 but not MIB1-C997S or MIB1-ΔRN3. Data are mean + SD (error bars). *, P < 0.05. (Student’s t test, n = 3). (D) Histogram of surface RYK expression on cells transfected with N-terminal glue-tagged RYKΔICD and with or without flag-MIB1. (E) Histogram of surface RYK expression on cells transfected with glue-RYK and control, MIB1, or RYK siRNAs. (F) Graph representing the number of cells in the RYK-positive gate shown in E. Data demonstrate that MIB1 loss of function significantly increases the level of RYK expression on the cell surface. Data are mean + SD (error bars). **, P < 0.005. (Student’s t test, n = 3).