β catenin ntd

Cells were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor tablets (Roche, IN, USA). Cell lysates were cleared by centrifugation and protein concentration determined by Bio-Rad Protein Assay kit (Bio-Rad Laboratories, CA, USA). 5–30 µg (as indicated in the figure legends) of total protein in SDS sample buffer was loaded per lane and separated on NuPAGE Tris-Acetate precast polyacrylamide gels (Life Technologies). Detection of proteins was done with the following primary Abs and dilutions: β-catenin CTD (BD Transduction Laboratories, #610153, 1∶2000), γ-catenin (BD Transduction Laboratories, #610253, 1∶5000) β-catenin NTD (Abcam, ab32572, 1∶5000), E-cadherin (BD Transduction Laboratories, #610181, 1∶4000), α-Catenin (BD Transduction Laboratories, #610193, 1∶500), p120-catenin (BD Transduction Laboratories, #610133, 1∶2000), Actin (Sigma-Aldrich, A2066, 1∶2000). Secondary Abs were: donkey anti-mouse IgG-HRP (Santa Cruz Biotechnology, CA, USA, sc-2314, 1∶5000), donkey anti-rabbit IgG-HRP (Santa Cruz biotechnology, sc-2313, 1∶5000). Signals were developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) and quantification of signal intensities was done using the Image Studio Lite software (LI-COR, NB, USA).

For immunostaining cells were fixed (4% paraformaldehyde) for 10 minutes at room temperature (RT) and blocked (5% bovine serum albumin (BSA), 5% goat serum, 0.1% Tween-20 in phosphate buffered saline (PBS)) for 1 hour at RT followed by incubation with primary antibody (Ab) (0.5% BSA, 0.5% goat serum, 0.1% Tween-20 in PBS) at 4°C over night. After washing in PBS cells were incubated with fluorescently labeled secondary Ab (0.5% BSA, 0.5% goat serum, 0.1% Tween-20 in PBS) for 30 minutes at RT. For nuclear counterstaining the cells were incubated with DAPI (1 µg/ml) for 10 minutes at RT. Cells stained on cover slips were mounted on slides using Fluorescence Mounting Medium (DAKO, Denmark). The following primary Abs and dilutions were used: β-catenin CTD (BD Transduction Laboratories, BD Biosciences, CA, USA, #610153, 1∶2000), γ-catenin (BD Transduction Laboratories, #610253, 1∶2000) β-catenin NTD (Abcam, Cambridge, UK, ab32572, 1∶250). Secondary Abs used were: Alexa Fluor 488 Donkey Anti-Mouse, (Life Technologies, A-21202, 1∶500), Alexa Fluor 488 Donkey Anti-Rabbit (Life Technologies, A-21206, 1∶500). Images were visualized under a fluorescence microscope (Zeiss Axiovert 200 M, Carl Zeiss, Germany) and acquired with CCD camera (Zeiss Axiocam HR).