Beads were washed twice with washing buffer (1/3 dilution of cell lysis buffer in phosphate-buffered saline (PBS)), and briefly centrifuged and resuspended in the sample buffer before being subjected to SDS-PAGE.
After centrifugation at 300 ×g for 5 min at 4°C, cells were then washed 1 2 times with cold staining buffer before being subjected to flow cytometry.
After centrifugation, the protein G-agarose pellets were washed several times with washing buffer (1/3 dilution of cell lysis buffer in PBS) and resuspended in sample buffer before being subjected to SDS-PAGE.
Protein concentration was determined by BCA assay (23223 and 23224; Thermo Scientific), and 10 20 µg protein was added to 2× Laemmli loading buffer before being subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and western blotting as described previously (50).
Gastric cancer cell lines including SGC-7901, BGC-823 and human normal gastric epithelium immortalized cell line GES-1 were cultured, collected and lysed with the RIPA buffer on ice before being subjected to Western blotting analysis.
Protein concentrations were determined by Bicinchoninic acid (BCA) protein assay reagent (Pierce) and lysates were boiled in a Laemmli samples buffer for 5 min before being subjected to SDS-PAGE.
For immunoprecipitation assays, cells were lysed in 0.2% Triton lysis buffer, and the resulting pellets were resuspended in NP-40 lysis buffer with 0.1% SDS before being subjected to sonication fifteen times (5 s each) at 200 W. Next, the lysates were incubated with the indicated antibodies overnight at 4°C and precipitated by protein G for 5 h.
Samples were washed three times 5 minutes in Co-IP buffer containing 100 mM NaCl before being subjected to 10% SDS-PAGE, followed by western blot.
Five hundred microliters of intact harvested cells were pelleted at 400×g for 5 min and resuspended in 100 μL of Reporter Lysis Buffer (Promega Corp., Madison, WI, USA), before being subjected to a freeze/thaw cycle.
To assess the activity of recombinant PTEN, 1 μg of PTEN was incubated with 1 μg of recombinant p85α in buffer containing 50 mM Tris HCl pH 8.0, 50 mM NaCl, and 10 mM MgCl2 (PTEN reaction buffer without DTT) at room temperature for 1 hr before being subjected to the reaction.
Paraffin sections were cut at 4 μm, dewaxed, and rehydrated before being subjected to heat-mediated antigen retrieval in a microwave using citrate buffer (10 m, pH 6.0).
