ABSTRACT
Objective To investigate the expression and association of Cx36 with ZO-1 in Cx36 transfected HeLa cells. Methods RT-PCR,PCR products cloning into PCR2.1TOPO vector,Cx36-pcDNA3 expression vector construction,cell culture,transient transfection using lipofectamine 2000,stable clone screening with G418,Western blotting,double immunofluorescence,and immunoprecipitation(IP) were used. Results Cx36-pcDNA3 expression vector was constructed and transfected into HeLa cells.Using homogenates from Cx36 transfected HeLa cells,Western blotting showed Cx36 band.By immunofluorescence microscopy,Cx36 transfected HeLa cells displayed punctate immunolabeling for Cx36 between cells.Irmmunolabeling for ZO-1 in these cells exhibited similar distribution to Cx36.By laser scanning confocal microscopy after double labeling with Cx36 and ZO-1 antibody revealed a high degree of Cx36 and ZO-1 colocalization at sites of cell-cell contact.Cx36 transfected HeLa cells were taken for IP with ZO-1 antibody,blots of IP protein probed with Cx36 antibody showed detectin of Cx36.Blots showed detection of ZO-1 after IP with Cx36 antibody from Cx36 transfected HeLa cells.Conclusion Cx36 was expressed in Cx36 transfected HeLa cells,Cx36 colocalizated and associated with ZO-1 in Cx36 transfected HeLa cells.