FIGURE 7.

Numb promotes Notch1 degradation. A and B, overexpression of Numb promotes Notch1 degradation not recycling. HEK293T cells expressing FL-Notch1 alone (A) or FL-Notch1 and Numb (B) were biotinylated, incubated at 18 °C for 2 h to accumulate biotin-labeled FL-Notch1 intracellularly, and then stripped of remaining surface biotin with MeSNa. Cells were then returned to 37 °C to resume trafficking. One set of cells was treated a second time with MeSNa followed by lysis to examine intracellular pools of biotinylated FL-Notch1 (IC, lower panels), whereas the second set was lysed without MeSNa treatment to examine total pools of biotinylated FL-Notch1 remaining (Surf + IC, upper panels). Pools of biotinylated FL-Notch1 were recovered with streptavidin beads and analyzed by Western blot using antisera specific for Notch1. Tfxn, transfection; IB, immunoblot. C and D, knockdown of Numb protein using Numb-specific RNA interference increases FL-Notch1 recycling back to the plasma membrane. C2C12 cells expressing a scrambled control siRNA duplex (C) or Numb-specific siRNA duplex (D) were analyzed as described above for intracellular and total pools of biotinylated FL-Notch1. Whole cell lysates were quantified and analyzed for Numb expression. Scram, scrambled siRNA; IC, intracellular; Surf + IC, surface and intracellular. Representative Western blots are shown. Films from three independent experiments were scanned and quantitated using an Alpha Innotech Fluorochem 8000 imaging system (see “Materials and Methods”), and results are shown in graphs (right). Data are expressed relative to total (Tot) surface-biotinylated Notch1 (mean ± S.D.; n = 3). Significance was determined by Student's t test; **, p < 0.01;*, p < 0.05 compared with respective controls shown in A and C.