Pcdna6 myc his b taglnag plasmid

Localization of endogenous transgelin in RKO, SW480, HCT116 and LOVO cell lines was determined by immuno uorescence. The primary antibody (anti-transgelin, 1:500, Abcam, USA), secondary antibody (Alexa Flour 594 goat anti-rabbit IgG, 1:500, Invitrogen, USA), and the VECTASHIELD mounting medium (Vector Laboratories, USA)) with 4′,6-diamidino-2-phenylindole (DAPI) were used. The immuno uorescence images were taken and preserved under the laser scanning confocal microscope (63× oil lens, Carl Zeiss, USA). Transfection SW480 and RKO cells were cultured in 12-well plates and transfected with pcDNA6/myc-His B-TAGLNag plasmid and pcDNA6/myc-His B-ag plasmid (Takara, Japan). In the validation experiment, we transfected the RKO cells with pENTER-TAGLN-Flag and pENTER-Flag control plasmid (Vigene Biosciences, USA). Transfection was conducted using Lipofectamine 2000/ Lipofectamine 3000 (Thermo Fisher Scienti c, USA). The cells were harvested at 48 hours after transfection for further analysis.

Localization of endogenous transgelin in RKO, SW480, HCT116 and LOVO cell lines was determined by immuno uorescence. The primary antibody (anti-transgelin, 1:500, Abcam, USA), secondary antibody (Alexa Flour 594 goat anti-rabbit IgG, 1:500, Invitrogen, USA), and the VECTASHIELD mounting medium (Vector Laboratories, USA)) with 4′,6-diamidino-2-phenylindole (DAPI) were used. The immuno uorescence images were taken and preserved under the laser scanning confocal microscope (63 × oil lens, Carl Zeiss, USA). Transfection SW480 and RKO cells were cultured in 12-well plates and transfected with pcDNA6/myc-His B-TAGLNag plasmid and pcDNA6/myc-His B-ag plasmid (Takara, Japan). In the validation experiment, we transfected the RKO cells with pENTER-TAGLN-Flag and pENTER-Flag control plasmid (Vigene Biosciences, USA). Transfection was conducted using Lipofectamine 2000/ Lipofectamine 3000 (Thermo Fisher Scienti c, USA). The cells were harvested at 48 hours after transfection for further analysis.