Mouse anti β catenin antibody

HaCaT cells were plated on 8-well slides and transfected and HDACi-treated as described already. At 24 hours post-transfection, cell preparations were rinsed with PBS and fixed with 4% paraformaldehyde (PAF) for 20 min. After several more washes with PBS and in PBS/0.01% triton X-100, non-specific sites on cells were blocked with a 20-times dilution of normal donkey antibody and the slides were left at room temperature for 1 hour. After this, a 100-fold dilution of mouse anti-β-catenin antibody (Santa Cruz Biotechnologies) was applied to the cells and slides were left to incubate in a humid chamber for 24 hours at 4°C. All further steps were carried out in the dark. Namely, after two washes in PBS, cell preparations were incubated in a 100-fold dilution of donkey-anti-mouse Cy3 (Vector laboratories) secondary antibody, for 30 minutes at room temperature. After that, washed labelled cells were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen). A week later, dried slides were visualized by fluorescence microscopy.

Subconfluent cells were washed twice with PBS, and then lysed with ice-cold RIPA lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 1 mmol/L EDTA, 1 mmol/L PMSF, and 1% cocktail of protease inhibitors) (pH7.4). The lysates were then clarified by centrifugating at 12,000 g for 20 min at 4°C. The protein extracts were separated by SDS-PAGE. The immunoblotting procedure was performed as described
[16 (link)] and the following antibodies were used: mouse anti-β-actin antibody (KangChen Bio-tech, Shanghai, China), rabbit anti-Akt antibody, rabbit anti-phospho-Akt (p-Ser473) antibody, rabbit anti-PI3K p85 antibody, rabbit anti-phospho-PI3K p85 (Tyr458) (Cell Signaling Technology, Danvers, MA), mouse anti-β-catenin antibody, rabbit anti-phospho-β-catenin (p-Ser33) antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized with ECL reagent (Thermo Scientific, Rockford, IL).