Anti beta catenin sc 7963

Non-confluent monolayers of EMT6 or 4T1 cells were incubated in L-CM or Wnt3a-CM, with or without 100 microgram/ml CS-E. Cells were fixed in 4% PFA, permeabilized using 0.2% TX-100, and immunofluorescence staining was performed according to standard methods. Primary antibody was anti-beta-catenin (sc-7963, Santa Cruz Biotechnology), followed by Alexa-488-conjugated secondary antibody (Invitrogen, USA), and DAPI as a nuclear stain.

Control and stimulated cells were washed twice with ice-cold PBS and scraped in RIPA lysis buffer (Sigma Aldrich). A mix of protease inhibitors (Complete-Mini Protease Inhibitor Cocktail Tablets, Roche, Mannheim, Germany) and phosphatase inhibitors (PhosStop; Roche, Mannheim, Germany) was added just before use. Cellular extracts were then centrifuged at 8000× g for 10 min. The Bradford assay was used to determine protein contents. For western blot analysis, cellular extracts were separated on SDS-polyacrylamide gels and proteins were blotted onto nitrocellulose membranes (BIO-RAD, Bio-Rad Laboratories, Hercules, CA, USA). The following antibodies were analyzed: anti-vinculin (7F9): sc-73614; anti-Rock1 (H-85): sc-5560 and anti-beta-catenin sc-7963 all from Santa Cruz Biotechnology; anti-p53 (acetyl k382) ab-75754 from Abcam; anti-TPT1 (E-AB-31729) from Elabscience; anti-E-cadherin (610181) from BD Bioscience. Antigens were detected with an enhanced chemiluminescence kit (Western Bright ECL HRP Substrate, Advansta Inc., Menlo Park, CA, USA), according to the manufacturer’s instructions.

The following primary antibodies were used in this study: anti-HMB-45 (1:20, Dako/ Agilent, Hamburg, Germany), anti-β-catenin (#9562, 1:50, Cell Signaling Technology, Leiden, Netherlands) and anti-Ki67/MIB1 (1:100, Dako/ Agilent). Immunohistochemistry of a human melanoma tissue microarray (TMA) using an anti-β-catenin antibody (1:100, Cell Signaling #9562) was performed as described previously [46 (link)]. For western blotting the following antibodies were used: anti-Phospho-Akt (Ser473) (#4060), anti-Akt (#9272), anti-PTEN (#9188), anti-beta-actin (#3700) (all Cell Signaling Technology) and anti-beta-catenin sc-7963 (Santa Cruz).
All work with the TMA and human material was approved by the local ethics committee (305/2017BO2).