Figure 3.

Cell-free synthesis of WNT proteins using Sf21 cell lysate. WNT proteins were synthesized in a batch-formatted Sf21 cell-free system. (A) Autoradiography of cell-free synthesized WNT proteins (WNT3a, WNT5a, WNT5b, WNT6). The qualitative analysis shows the expected molecular weight of the produced proteins. Production of WNT types was carried out with a human signal peptide (on the basis of pcDNA3.1(+)-WNT) and with a melittin signal peptide (on the basis of Expression PCR product). Translation mixture (TM) of cell-free synthesized WNT proteins were separated by centrifugation into a supernatant fraction (SN) and microsomal fraction (MF). (B) Analysis of translocation of WNT-eYFP fusion proteins by confocal laser scanning microscopy. (C) Analysis of Mel-WNT3a protein yield in a time course experiment. Radiolabeled proteins were TCA precipitated, transferred to a filter paper and dissolved in scintillation cocktail. Protein yield was determined by scintillation measurement using an LS6500 multi-purpose scintillation counter (PerkinElmer). (D) Schematic overview of the assay for functional characterization of WNT proteins based on canonical WNT signaling. (E) Functional characterization of cell-free synthesized Mel-WNT3a (50 ng/µL and 100 ng/µL) using the previously illustrated assay. Western blot analysis of accumulated β-catenin using primary anti-β-catenin and secondary anti-rabbit-HRP antibody (Cell Signaling). Protein band was detected using an ECL reagent (Promega) and a Typhoon Trio Plus Imager (GE Healthcare). Image analysis was performed with a housekeeping gene (β-actin) for normalization using ImageQuant TL software (GE Healthcare). NTC: No template control (cell-free reaction without WNT DNA template).