Figure 4.

Intracellular antibody LL3 and CDR3 mutant control intracellular antibody LL3.CDRAAA. (A) CDR3 alignments of LL3 and LL3.CDRAAA. (B) β-Catenin binding ELISA results for LL3 and LL3.CDRAAA with scFc tags. Biotinylated β-catenin was captured on to streptavidin coated plates, and VHH-scFc constructs were then added in half log dilutions from 32 nM and revealed with anti-mouse Fc HRP. The plates were read at 630 and 490 nm and ΔOD recorded. The negative control is a vector only DNA control transfection culture supernatant. Error bars represent 95% confidence limit. (C) Intracellular expression of intracellular antibodies LL3 and LL3.CDRAAA. Expression vectors containing VHH intracellular antibody encoding genes were transfected into HEK293 luciferase bioassay cells, with WNT1-containing vector. Forty hours post transfection, cells were resuspended in LDS sample buffer, 10 μL of each sample was run on a 4–12% bis-tris gel followed by Western blot, probed with mouse anti-myc. Analysis of band density was performed on GE Healthcare ImageQuant analysis software. (D) Relative activities of intracellular antibodies LL3 and LL3.CDRAAA in the HEK293 Wnt-induced luciferase reporter bioassay. LL3 and LL3.CDRAAA were tested for firefly and Renilla luciferase activity in the bioassay. Wnt signaling was induced by co-transfection of the Wnt1 gene. Results plotted as firefly luciferase activity relative to Renilla luciferase activity for each well. All conditions performed in 5 replicates. Data shows mean and SEM from a representative experiment. **** denotes p ≤ 0.0001, *** denotes p of 0.001-0.00011, for a 2 tailed ratio paired Student's t-test.