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目的探讨ECRG4在脂筏中分布及与细胞膜胆固醇含量的相关性。方法通过RT-PCR及Westernblot鉴定ECRG4阳性表达细胞株,用甲基-β-环糊精去除细胞膜上的胆固醇,超速离心的方法分离细胞膜,Western blot检测ECRG4蛋白含量变化。密度梯度离心制备脂筏,通过Western blot检测ECRG4蛋白水平。结果ECRG4在DU4475细胞中高表达,降低细胞膜上胆固醇后,ECRG4水平未发生明显变化;同时Western blot结果证实ECRG4未分布于脂筏。结论 ECRG4蛋白未定位于脂筏,且与细胞膜胆固醇之间无相关性。
Objective To investigate the distribution of ECRG4 in lipid rafts and the correlation with ECM cholesterol content. Methods ECRG4 positive cells were identified by RT-PCR and Western blot. Cell membrane was removed by methyl-β-cyclodextrin and the membrane was separated by ultracentrifugation. The content of ECRG4 protein was detected by Western blot. Lipid rafts were prepared by density gradient centrifugation and ECRG4 protein levels were determined by Western blot. Results ECRG4 was overexpressed in DU4475 cells and the level of ECRG4 did not change significantly after the reduction of cholesterol in the plasma membrane. Western blot analysis showed that ECRG4 was not distributed in lipid rafts. Conclusion ECRG4 protein is not located in lipid rafts and has no correlation with cell membrane cholesterol.