Figure 2
E3 ubiquitin ligases Hrd1 and Gp78 were involved in the degradation of CD147(N152Q)-EGFP.
(a) The effect of proteasome inhibitor (MG-132) on CD147(WT)-EGFP and CD147(N152Q)-EGFP. K7721 cells stably expressing CD147(WT)-EGFP and CD147(N152Q)-EGFP were cultured for 24 h in the presence or absence of 10 μM MG-132. The cell lysates were immunoblotted with the indicated antibodies. (b) The effect of lysosomal inhibitor (chloroquine) on the CD147(WT)-EGFP and CD147(N152Q)-EGFP. K7721 cells stably expressing CD147(WT)-EGFP and CD147(N152Q)-EGFP were cultured for 16 h in the presence or absence of 50 μM chloroquine. The immunoblots of cell lysates were probed with the indicated antibodies. (c) Both CD147(WT)-EGFP and CD147(N152Q)-EGFP were ubiquitinated. Extracts prepared from K7721 cells stably expressing CD147(WT)-EGFP or CD147(N152Q)-EGFP were immunoprecipitated (IP) using an anti-EGFP antibody, and the resulting precipitates were examined by immunoblot analysis using the indicated antibodies. (d) Knockdown of Hrd1 increased the protein levels of CD147(WT)-EGFP and CD147(N152Q)-EGFP. K7721 cells stably expressing CD147(WT)-EGFP or CD147(N152Q)-EGFP were transfected with control or Hrd1 siRNAs for 3 days. Immunoblots of cell lysates were probed with the indicated antibodies. Relative expression of CD147-EGFP/Tubulin were quantified using Image J software, *P < 0.05, **P < 0.01. (e) Knockdown of Gp78 increased the protein level of CD147(N152Q)-EGFP. K7721 cells stably expressing CD147(WT)-EGFP or CD147(N152Q)-EGFP were transfected with control or Gp78 siRNA for 3 days. Immunoblots of cell lysates were probed with the indicated antibodies. Relative expression of CD147-EGFP/Tubulin were quantified using Image J software, no statistical significance (NS), **P < 0.01. (f,g) CD147(N152Q)-EGFP co-immunoprecipitated with Hrd1 and Gp78. Extracts prepared from K7721 cells stably expressing CD147(N152Q)-EGFP were coimmunoprecipitated using the indicated antibodies. The resulting precipitates were examined by immunoblot analysis using the indicated antibodies. (h) Additive effect of Hrd1 and Gp78 co-inactivation on CD147(N152Q)-EGFP. K7721 cells stably expressing CD147(N152Q)-EGFP were transfected with control siRNA, or siRNA against Hrd1 (#2), Gp78 (#2), or both for 3 days. Lysates prepared from these cells were immunoblotted using the indicated antibodies. Relative expression of CD147-EGFP/Tubulin were quantified using Image J software, *P < 0.05, **P < 0.01.
