Curix 60 processor

Chemiluminescence was detected with a photographic film (Amersham Hyperfilm ECL, GE Healthcare, Chalfont St. Giles, UK) in a Curix 60 processor (Agfa, Mortsel, Belgium). Films of immunoblots were scanned into TIF format using a CanoScan LiDE 35 (Canon, Tokyo, Japan) and digital images were imported and quantified using ImageJ software (National Institutes of Health, Berthesda, MD, USA) following the method outlined in [33 ]. The quantitative band intensities of Wnt3a mutant proteins were normalized relative to the wild-type Wnt3a protein band intensity.

Cells were trypsinized, washed with PBS⁻, and lysed in IP or RIPAS buffer supplemented with protease inhibitor cocktail (Roche). The lysates were incubated for 15 min on ice, centrifuged, and frozen. Immunoblotting was performed using aliquots (15–30 μg) of the whole-cell extract. Proteins were separated on polyacrylamide gels (8 or 12%) and blotted onto PVDF (Immobilon-P Transfer Membrane; Millipore) or nitrocellulose (Amersham Protran Premium) membranes. Antibodies used for detection are listed in Supplementary material (Table S3). The chemiluminescent substrate signal (Thermo Scientific; 34079, 34095) was developed using the Curix60 processor (Agfa).

Adherent cells were scraped and tissues were homogenized in cold RIPA lysis buffer (R0278, Merck) supplemented with protease and phosphatase inhibitors using an IKA T8 Ultra Turrax tissue homogenizer (Cole-Parmer). Protein concentration was determined using the Pierce BCA Protein Assay (23225, Thermo Scientific) following the manufacturer’s instructions. Total protein extract (12–45 µg) was supplemented with 5X loading buffer (4% glycerol, 0.5 M Tris-HCl pH 6.8, 8% SDS, 0.04% bromophenol blue, 5% β-mercaptoethanol) and resolved on Any kD Criterion TGX Precast Stain-free gels (5678124, Bio-Rad). Proteins were transferred to 0.2 µm PVDF membranes using a TransBlot Turbo Transfer System (Bio-Rad) and total protein was quantified using Ponceau S staining (78376, Merck). Membranes were blocked with 5% non-fat milk in PBS-T (0.5% Tween-20 [1706531, Bio-Rad] in PBS) for 1 h. Membranes were then incubated overnight at 4 °C in primary antibodies diluted 1:1000 (Supplementary Table 1) in 5% BSA in PBS, and subsequently for 1 h at room temperature in secondary antibodies diluted 1:2000–1:4000 in PBS-T. Membranes were developed using Pierce ECL Western Blotting substrate (32106, Thermo Fisher) or Amersham ECL Prime (10308449, Cytiva) and x-ray film (AGFA) using a CURIX 60 Processor (AGFA).