Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

Figure 1 Electrophoresis analysis of RT-PCR products of MKS and MKN genes. L1: β-actin (positive control); L2: fragment of MKS; L4: fragment of MKN; L3 and 5: 100 bp DNA ladder.

Figure 2 Identification of constructed plasmids by digestion with restriction endonucleases (EcoRI and BamHI). L1: 1000 bp marker; L2: pEGFP-N2; L3: pEGFP-MKS; L4: pEGFP-MKN; L5: 100 bp marker.

Figure 3 Western blotting analysis of MK-GFP expressed in HepG2 cells transfected with various plasmids. Cells were transfected with pEGFP-MKS (L1), pEGFP-MKN (L2), pEGFP-N2 (L3) and with no plasmids (L4).

Figure 4 Presence of MK in the nucleoli of HepG2 cells. The cells were stained with goat anti-MK antibody (1:1000). Cells were fixed with PFA and the secondary antibody FITC-conjugated mouse anti-goat antibody was added. The staining (green for MK) is presented (× 400).

Figure 5 Dynamic imaging of the subcellular localization of MK-GFP fusion protein in HepG2 cells captured by laser scanning confocal microscopy (marked by an arrow). A-F: 11 h 0 min, 11h 30 min, 12 h, 12 h 40 min, 15 h 40 min, 16 h 40 min after transfection, respectively.

Figure 6 Subcellular localization analysis of MKS-GFP and MKN-GFP fusion proteins in carcinoma cell lines. Twenty-four hours after transfection HepG2 cells (upper pannel)and DU145 cells (bottom pannel) were separately transfected with EGFP-MKS and EGFP-MKN. MKS-GFP and MKN-GFP fusion proteins were localized to nucleus and accumulated in nucleolus, but rarely localized to cytoplasm. The high light “spots” in the center of nuclei are nucleoli. HepG2 cells (upper panel) and DU145 cells (bottom panel) were also transfected with pEGFP-N2 for control. It seemed that GFP protein could diffuse freely in the whole cell except the nucleoli. The whole cell is light and the low light “spots” are nucleoli.