FIGURE 3.

Knockdown of Sel1L or Hrd1 inhibits NHK dislocation. A, HeLa cells stably expressing SP-S11-NHK-HA and S1-10 were transfected with control siRNA or siRNA targeting Sel1L or Hrd1. Forty-eight hours after transfection, the cells were treated with MG132 (10 μm) for 4 h before being imaged with fluorescent microscope. B, cells were transfected as in A. Forty-eight hours after transfection, the cell culture medium was replaced with phenol red-free medium containing MG132 (10 μm). The drGFP images were acquired every 10 min for up to 4 h. C, measurement of drGFP intensity in B using ImageJ. The data are presented as the means ± S.E. of at least 30 cells. D, cells were transfected as in A. Forty-eight hours after transfection, the microsomes from these cells were prepared and treated with proteinase K (100 μg/ml) as indicated for 20 min on ice. Then, the microsomes were lysed and processed for IB. E, cells transfected as in A were processed for immunoprecipitation (IP) with an anti-HA antibody followed by IB with an anti-ubiquitin antibody to detect polyubiquitinated S11-NHK. The numbers in the polyubiquitin blot (PolyUb, lanes 4–6) show the relative density of polyubiquitinated S11-NHK-HA. WCL: whole cell lysate. F, HeLa cells stably expressing SP-S11-NHK-HA and S1-10 were subject to siRNA knockdown of Sel1L, Hrd1, gp78, or p97/VCP. NC: non-targeting control siRNA. Graph shows the relative drGFP fluorescence intensity in the knockdown cells measured on a fluorescence microplate reader. Blots show the efficiency of gp78 and p97/VCP knockdown and the levels of NHK.