Stericup quick release

Recombinant STA, RA, biotinylated IDR proteins, and LAF-fused Dronpa proteins were all prepared as previously described^{24 (link)}. Briefly, biotinylated IDR proteins were expressed in AVB101 (Avidity) cells, and other proteins were expressed in Escherichia coli BL21 (DE3). All proteins were purified by Ni-IDA columns (BioProgen, Daejeon, South Korea). Avidin proteins were produced as inclusion bodies, which were dissolved in 6 M Guanidine hydrochloride (GuHCl) and 50 mM Tris-HCl pH 8.0 for overnight at 4 °C. For refolding, denatured STA and RA proteins were diluted dropwise into PBS and filtered through a 0.22 μm membrane filter (Stericup® Quick Release, Millipore Express® PLUS), followed by overnight incubation at 4 °C before column purification. IDR-fused proteins were stored in a buffer containing 200 mM NaCl, 50 mM Tris pH 8.0, and 10% Glycerol. 1.0 LAF-Dronpa and 0.5 C LAF-Dronpa were labeled with NHS-Cy5 (Lumiprobe) by mixing proteins with a dye in a 1 : 0.5 protein/dye ratio. The mixed solutions were incubated for 40 min at 25 °C with shaking, and Cy5-labeled LAF-Dronpa was purified by a PD10 desalting column (Sephadex™ G-25 M, GE Healthcare). Protein concentrations were determined by Bradford assays and OD_280 nm measurements with the Beer–Lambert equation.

Wnt-3α-expressing cells were purchased from ATCC (CRL-2647). Cells were cultured in Greiner Bio-One CELLCOAT™ tissue culture-treated T-75 flasks at an initial concentration of 1.5 × 10⁶ cells. The cells were cultured in 20 mL of Wnt-3α growth medium (WGM) (Supplementary Table S1) and passaged once 75% confluency was reached. We used 0.4 mg/mL G-418 for selection and cells were kept growing on selection medium for a week before producing conditioned medium. The cells were lifted using 3 mL of TrypLE (Life Technologies, Carlsbad, CA, USA) and passaged. The process was repeated until approximately 40–50 T75 flasks were generated. The media were replaced with 100 mL of harvest medium (HM) (Supplementary Table S1). The cells were then incubated in HM for one week without media changes. After one week, the media were collected, and tubes were centrifuged at 500× g for 5 min at 8 °C. Following this step, the media were filtered using filter cups (Stericup Quick Release, Millipore, Burlington, MA, USA). Lastly, 25 mL of the collected harvested medium was separated into 50 mL canonical tubes and utilized fresh or stored for no more than 2 weeks at 4 °C. Recombinant human Wnt-3α (R&D, 5036-WN010) was used at a final conc. of 100 ng/mL as a positive control, and Wnt activity of conditioned medium could be examined by the TOP/FOP assay as described [49 (link)].