Figure 5
The first 100 amino acids of CT105 contain a Golgi-targeting region. HeLa cells were transfected for 24 h with plasmids encoding monomeric EGFP (mEGFP) or different regions of CT105 fused to the C-terminus of mEGFP (CT105-mEGFP proteins), as indicated. (A) Schematic representation of the mEGFP-CT105 proteins expressed and analyzed. (B) Total extracts of transfected cells were analyzed by immunoblotting with antibodies against GFP and α-tubulin (HeLa loading control) using SuperSignal West Pico detection kit (Thermo Fisher Scientific). (C) Transfected cells were fixed with 4% (w/v) PFA, labeled with antibodies against TGN46 (red), and the appropriate fluorophore-conjugated secondary antibody, and imaged by confocal fluorescence microscopy. Images correspond to single z sections. Scale bars, 10 µm. (D) Cells immunolabeled as described in C were enumerated by fluorescence microscopy for localization of mEGFP-CT105 only at the Golgi, only at the plasma membrane (PM), or both at the Golgi and plasma membrane (Golgi + PM). Data are mean ± standard error of the mean of three independent experiments (n = 100). (E) Cells were treated for 1 h with DMSO or 1 µg/ml BFA. Then, the cells were either fixed with 4% (w/v) PFA (upper and middle panels) or washed with complete medium lacking BFA and incubated for an additional 1 h and then fixed with 4% (w/v) PFA (lower panels). The fixed cells were immunolabeled with antibodies against TGN46 (red), and the appropriate fluorophore-conjugated secondary antibody, and examined by confocal immunofluorescence microscopy. Images correspond to single z sections. In the area delimited by a white square (left-side panels) images were zoomed (right-side panels). Scale bars, 10 µm.
