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. 2019 Jan 15;11(1):96. doi: 10.3390/cancers11010096

Figure 1.

Figure 1

Design of DNA constructs and expression in HEK-293 cells: (A) Schematic of DNA constructs used in this study. This scheme shows the arrangement of streptavidin (SA), double mutant form of HPV-16 E7, and the extracellular domain of mouse 4-1BBL. A signal peptide (SP) was fused to the N-terminus of the recombinant genes. The controls were CRT-E7wt and empty plasmid. (B) A representative image of western blot analysis. 5 × 105 HEK-293 cells were transfected with constructs expressing CRT-E7wt, SP-SA-E7-4-1BBL, SP-SA-4-1BBL, and empty vector as negative control. Twenty-four hours later, cells were lysed, and protein expression was analyzed by western blot using E7 antibody. (C) Immunofluorescent staining of transfected HEK-293 cells. HEK-293 cells were transfected with constructs expressing CRT-E7wt, SP-SA-E7-4-1BBL, SP-SA-4-1BBL, and empty vector as negative control. Twenty-four hours later, cell slides were fixed and incubated with E7 and 4-1BBL antibodies, and the specific secondary antibodies conjugated with fluorochromes. Evaluation of DNA construct expression was performed by fluorescence microscopy. Images were taken at 40× magnification, DAPI was used as a marker for nuclei. DAPI, 4′,6-diamidino-2-phenylindole; CMV, cytomegalovirus promoter; CRT, calreticulin; SP, signal peptide.