Figure 5.

Stability and trafficking from ER to the plasma membrane. In (A), transiently transfected HEK293 cells were cultured in the presence of cycloheximide (20 μg/ml) for 0, 2, 4, and 6 h. Then, cells were lysed and analyzed by western blot using anti FLAG-antibody³³. Anti actin is used as loading control³³. The western blot image is representative of three independent experiments. The histogram represents mean of values obtained by scanning densitometry of immunoblots; the quantification was performed for WT or Qm comparing each time to the control (time = 0). Data from all experiments are shown in Supplementary Table S1. Significantly differences were estimated by Student's t-test (a = 3.7E−07, b = 0.00211, c = 2.9E−06, d = 0.00036 and e = 0.000537). In (B), HEK293 cells transiently transfected with WT or Qm constructs were treated with 5 μM BFA for 7 h. The treatment blocks the protein trafficking toward the plasma membrane allowing for the retention of CD98 into the ER. The rescue of the protein trafficking was obtained by washing out BFA. Cells were fixed and labeled at the indicated times. Confocal images showed a single, representative, section of a Z-series taken through the entire cell. Scale bar 10 µm.