Anti non phospho active β catenin

Tissues and cells were lysed in cold RIPA buffer containing a proteinase inhibitor Phenylmethanesulfonyl fluoride (Beyotime cat#ST506) and a phosphatase inhibitor (Thermo scientific Inc. cat#78420). The protein concentrations were determined by the BCA protein assay (Thermo Scientific Inc. cat#23227). The samples were adjusted to equal protein concentrations and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins on the gel were transferred onto PVDF membranes, and then were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature. The membranes were incubated with the anti-PCDH18 (Santa Cruz cat#sc-104574), anti-nonphospho (active)-β-catenin, anti-phospho-β-catenin, anti-β-catenin, anti-phospho-GSK-3β, anti-GSK-3β, anti-caspase3 (Cell Signaling Technology cat#8814, 4176, 8480, 9331, 9315, 9662), anti-p21, anti-cyclin A1, anti-cyclin D1, anti-cyclin E1 (Bioworld cat#AP0713, BS1084, BS2436, BS1085) and anti-GAPDH (Kangchen cat#KC5G4) overnight at 4 °C. After being washed with Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO). The immunoreactive bands were detected with ECL kit (Lu long Inc. cat#ECL-100-ES) and cropped from Supplementary Fig. 2. Each sample was tested in duplicate.

Cells were fixed in 2% paraformaldehyde at room temperature (RT) for 20 min. After blocking and permeabilizing with 1% BSA and 0,3% Tryton X-100 for 1 h at RT, cells were incubated with primary antibody overnight at 4 °C. Dilutions of primary antibodies were in accordance with the manufacturer’s instructions. Cells were then incubated in the dark at RT for 1 h with corresponding secondary antibodies conjugated to Alexa Fluor plus 555 or Alexa Fluor 488 (Thermo Fisher Scientific). Lastly, nuclei were stained with Hoechst 33,342 (Thermo Fisher Scientifc) and the coverslips were mounted on the slides with Fluoromount-G (Thermo Fisher Scientific). Slides were then examined under a Zeiss AxioImager Z1 fluorescent microscope (Carl Zeiss International, Germany). Different fields were examined at 40× and 63× magnification. The following antibodies were used: anti-PLCβ1 (Thermo Fisher Scientific) diluted 1:100, anti-Ki67 (Cell Signaling Technology) diluted 1:400, anti-Non-phospho (Active) β-Catenin (Cell Signaling Technology) diluted 1:600.

WNT5A-expressing breast cancer cells or parental breast cancer cells treated with PFKP siRNA, rWNT5A, XAV939, U0126 or not were washed with ice-cold PBS and lysed in ice-cold phosphorylation lysis buffer (PLB). The protein estimation, SDS-PAGE and visualization procedures were performed as described in Prasad et al. [40 (link)]. The following primary antibodies were used: anti-WNT5A from R&D systems (MN, USA); anti-Hexokinase-II, anti-Puruvate Kinase; anti-PKFP, anti-non-phospho (Active) β-catenin, and anti-pERK1/2 antibodies from Cell Signaling Technology (MA, USA); anti-MCT1 antibody from Santa Cruz Biotechnology Inc. (TX, USA); and anti-β-actin antibody from Sigma-Aldrich (MO, USA). The secondary antibodies used were goat anti-mouse, goat anti-rabbit and rabbit anti-goat, which were procured from Dako (Glostrup, Denmark). Separated protein bands were visualized using Chemiluminescence HRP substrate (Millipore), and the membranes were imaged and analyzed using the Chemi Doc™ imaging system from Bio-Rad.

For WB, cells were collected and lysed in RIPA buffer (Cell Signaling) with a complete protease inhibitor cocktail (Roche), phosphatase inhibitors (Roche), and PMSF (Sigma), then centrifuged at 12,000g for 10 min at 4 °C. Protein concentration was measured by Bio-Rad protein assay kit (Hercules, CA) according to the manufacturer’s instructions. Western blot assays were performed as previously described^{12 (link)}. The primary antibodies were obtained as follows: anti-caspase-3, anti-PCNA, anti-β-Catenin, anti- Non-phospho (Active) β-Catenin, and Exosomal Marker Antibody Sampler Kit antibody (Flotillin-1 and EpCAM) from Cell Signaling Technology, anti-β-actin antibody from Abmart.