Correlating expression levels of recombinant N by ELISA and Western blot with sdAb-mNeonGreen cell staining. (A) Schematic of the expression cassettes of the vectors used to elevate recombinant N protein levels for sdAb-mNeonGreen mediated detection in HEK293T cells. hCMVie, human cytomegalovirus promoter-enhancer; BGH, bovine growth hormone polyadenylation signals; AdTri-IVS, adenovirus tripartite leader with intervening sequence; CMV intron A, cytomegalovirus intron A; mutant HPREzb, woodchuck hepatitis virus post-transcriptional regulatory element. (B) ELISA titration of RIPA lysates from transient expressions of N protein, or control β-gal, captured using oriented sdAb as captor and phage displayed sdAb as tracer. The transfections and sandwich assay were performed twice and plots are the mean with error bars representing +/− SD. (C) Western blotting of the RIPA lysates of the pcDNA or puma1-5 vectors expressing WuN, or β-gal control, probed with sdAb-APEX2 and developed with SuperSignal West Pico chemiluminescent substrate. Molecular weight markers are indicated in kDa. (D) Transient transfections of control, parental pcDNA or puma1-5 vectors probed with 100 nM of the Wu3 sdAb-mNeonGreen fusion protein 24 h post-transfection (10x magnification).