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. 2017 Sep 27;8:1197. doi: 10.3389/fimmu.2017.01197

Figure 3.

Figure 3

Evaluating expression of various single-domain antibody (sdAb) formats as intrabodies within HEK 293T cells. (A) Mammalian expression vector puma1 built for these studies utilizes a human cytomegalovirus immediate early gene enhancer and promoter (CMV IE) and the adenovirus tripartite 5′-non-coding region with a hybrid splice donor acceptor with other components from the pcDNA stable (Invitrogen) including high copy number in both Escherichia coli and HEK 293T cells and G418 resistance cassette for stable line generation. (B) Anti-C9 Western blot of whole cell lysates of HEK 293T cells transiently transfected with C9 tagged wild-type llama sdAb sequences or human optimized sequences identify those clones with low and high relative expression levels. Cells were harvested 72 h post-transfection. (C) Anti-C9 Western blot of HEK 293T soluble (including cytosol) and insoluble (including membranes) fractions following transient expression of llama sdAb as monomeric and dimeric versions. (D) Coomassie blue stained SDS-PAGE of the soluble fractions reveal visible bands at the expected places for sdAb B and E monomers and just visible are the corresponding dimers.