Expression and localization of p21-activated kinase 4 in gastric cancer cells.

Objective To construct an eukaryotic expression vector pEGFP-PAK4 and identify the expression and localization of extraneous and endogenous PAK4 in gastric cancer cells.Methods Using pCAN2-MYC-PAK4 as template,we obtained human PAK4 coding sequence by PCR amplification and cloned it into the eukaryotic expression vector pEGFP-C1.BGC-823 cells were transiently transfected with plasmid pCAN2-MYC-PAK4 and the expression of MYC-PAK4 fusion protein was analyzed by Western blot.The expression of endogenous PAK4 in different gastric cancer cells was also examined by Western blot.pEGFP-PAK4 was then transiently transfected into gastric cancer BGC-823 cells to determine the subcellular localization of PAK4 by laser scanning confocal microscope.Results(1)The coding sequence of hPAK4 was cloned into pEGFP-C1 eukaryotic expression vector;(2)The expression of MYC-PAK4 fusion protein and endogenous PAK4 in gastric cancer cells were identified;(3)Green fluorescence was observed in the cytoplasm of gastric cancer BGC-823 cells after transfection of pEGFP-PAK4 plasmid.Conclusion The plasmid pEGFP-PAK4 was successfully constructed.The expression of extraneous and endogenous PAK4 was identified.PAK4 was mainly localized in the cytoplasm of gastric cancer BGC-823 cells.This study provides the basis for research on biological features and signal transduction pathways of PAK4.