Ripa extraction reagent

The detection of WWOX protein expression in the cell lines was carried by the use of Western blot. Briefly, total protein was extracted and separated in 10% SDS‐PAGE, and then transferred to PVDF membranes. The lysis buffer was RIPA extraction reagent (Pierce Biotechnology, Rockford, IL, USA. Cat No#89900), containing phenylmethylsulfonyl fluoride. In addition, we loaded 20 μg of total protein per well for blotting. Pore size of the PVDF membrane was 0.45 μm (Immobion‐P Transfer Membrane, Millipore, Billerica, MA, USA. Cat No# IPVH 00010). The membranes were then washed, blocked with 5% nonfat dry milk, and followed by incubation with specific primary antihuman antibodies against WWOX (1:1000; Abcam, Cambridge, UK. Cat No# ab189410), GAPDH (1:1000; Sigma‐Aldrich, USA. Cat No# G9295) at 4°C overnight, and secondary appropriate antibody (Peroxidase‐conjugated AffiniPure Goat Anti‐Rabbit/Mouse IgG (H+L), 1:5000; Jackson ImmunoResearch, USA. Cat No#111‐035‐003; 115‐035‐003) for 1 hour at room temperature on the next day. Enhanced chemiluminescence assay (Pierce Biotechnology, Rockfield, IL, USA. Cat No#32209) was used to visualize the bands, and the blotted protein bands were exposed to Tanon‐5200 Chemiluminescent Imaging System (Tanon, China). Protein levels were quantified by ImageJ software.

Immunoblotting was performed as described previously.^{40 (link)} In brief, protein was isolated from 70 to 80% confluent cultured cells using RIPA Extraction Reagent (Pierce Biotechnology, Rockfield, IL, USA) following the manufacturer's directions. Equal amounts of protein were resolved on 4–20% SDS polyacrylamide gels and transferred to nitrocellulose membrane. The resulting blots were blocked with 5% non-fat dry milk and probed with antibodies. All antibodies were obtained from Cell Signalling Technology Inc. Denver, MA, USA except RUNX2 and GAPDH which were purchased from Santa Cruz Biotech. Blots were visualized using Western blotting luminal reagent (sc-2048; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Cells were lysed with RIPA extraction reagent (Thermo Fisher Scientific, MA, USA) to obtain total protein. Total protein samples (40 μg) were separated by SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Then, the PVDF membranes were incubated with primary antibodies against PTEN, total AKT, phosphorylated AKT (p‐AKT) and GAPDH (Cell Signaling Technology, Danvers, MA, USA). Bands were visualized by detection with the ECL substrate.