Figure 3

CD147(N152Q)-EGFP was more associated with the heat shock proteins than with the ER lectin chaperones.

(a) The localizations of CD147-EGFP and its binding partners, CNX, CRT, GRP78, and GRP94, were determined by immunofluorescence staining in K7721 cells stably expressing CD147(WT)-EGFP and CD147(N152Q)-EGFP. Scale bars, 20 μm and 10 μm. (b) The interaction between CD147 and the chaperones. Extracts prepared from K7721 cells stably expressing CD147(WT)-EGFP or CD147(N152Q)-EGFP were immunoprecipitated using the indicated antibodies. The resulting precipitates were examined by immunoblot analysis using the indicated antibodies. The signal intensities of each chaperone and CD147-EGFP were quantified using Image J software and are presented as the ratio of chaperone/CD147-EGFP signal. *P < 0.05, **P < 0.01, and **P < 0.001. (c) The effect of CNX knockdown on the binding ability among CD147(N152Q)-EGFP, GRP78, and GRP94. K7721 cells stably expressing CD147(N152Q)-EGFP were transfected with control or CNX siRNA for 3 days, followed by immunoprecipitated with the indicated antibodies, **P < 0.01. (d) The effect of GRP78 knockdown on the binding ability among CD147(N152Q)-EGFP, CNX, and GRP94. K7721 cells stably expressing CD147(N152Q)-EGFP were transfected with control or CNX siRNAs for 3 days, followed by immunoprecipitated with the indicated antibodies, **P < 0.01. (e) Expression of CNX, CRT, GRP78, and GRP94. Lysates of K7721 cells stably expressing the CD147(WT)-EGFP and CD147(N152Q)-EGFP mutants were analysed by Western blotting with the indicated antibodies.