Anti myc sc 789

The cells were lysed with RIPA buffer (Millipore, Billerica, MA, USA) containing protease inhibitor cocktail (Roche), and the lysates were quantified with a protein assay kit (Bio-Rad, Hercules, CA, USA). The cell lysates were separated using SDS-PAGE and then transferred to PVDF membranes. The proteins were identified while using appropriate antibodies. Anti-VGLL1 (10124-2-AP) was purchased from Proteintech (Rosemont, IL, USA). Anti-TEAD4 (ab58310) and anti-MMP9 (ab76003) were purchased from Abcam (Cambrige, MA, USA). Anti-GFP (NB600-308) was purchased from Novus Biologicals (Centennial, CO, USA). Anti-Myc (sc-789) and anti-HA (sc-805) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-GAPDH (LF-PA0212) was purchased from AbFrontier (Seoul, Korea). Anti-β-tubulin (2128), anti-pSer473-AKT (9271), anti-AKT (9272), anti-p-β-catenin (9561), and anti-β-catenin (9562) were purchased from Cell Signaling (Danvers, MA, USA). Anti-Flag (F1804) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The protein signal was detected while using an enhanced chemiluminescence kit (Millipore, Burlington, MA, USA).

At 48 h post-transfection, NIH 3T3 cells or HEK293 cells were harvested and lysed in Pierce IP lysis buffer (87787, ThermoFisher Scientific). The lysates were centrifuged for 10 min at 13,000 r.p.m. at 4°C. One milligram of lysate was incubated with specific antibodies [e.g. anti-HA monoclonal antibody (sc-7392, Santa Cruz Biotechnology) or mouse IgG (I5381, Sigma-Aldrich)] overnight at 4°C with continuous end-over-end mixing. Thirty microliters of protein-G–agarose (11719416001, Roche, Switzerland) beads were added to the samples and incubated for a further 2 h at 4°C with continuous end-over-end mixing. Samples were then washed once with Pierce IP buffer and then with Tris-buffered saline (TBS) before being denatured in SDS sample buffer for 7 min at 95°C. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes (HATF00010, Millipore). Membranes were blocked with 5% non-fat dry milk for 1 h and then incubated with anti-myc (sc-789, Santa Cruz Biotechnology), anti-HA (sc-805, Santa Cruz Biotechnology) or anti-GFP (Invitrogen) polyclonal antibodies for 2 h at room temperature. After being washed three times in 0.1% TBST, membranes were incubated for 1 h at room temperature with anti-rabbit HRP secondary antibody (A9169, Sigma-Aldrich). Results were visualized by the Chemidoc MP imaging system (Bio-Rad).