F1804

One day after transfection, HEK293T cells were collected and resuspended by a lysis buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 1× protease inhibitor cocktail (TargetMol, C001), 1× phosphatase inhibitor cocktail (MedChem Express, HY-K002 & HY-K0023), 1% Triton X-100. Sodium dodecyl sulfate (SDS) loading buffer was immediately added to the cell lysate. Samples were boiled for 15 min, separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). The membranes were subsequently blocked with 3% BSA in TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) for 1 h. The PVDF membranes were immunoblotted with antibodies against pTyr(PY99) (Santa Cruz, SC7020, dilution 1:100), GFP (TransGen, HT801, dilution 1:3000), Flag (Sigma, F1804, dilution 1:3000) or GAPDH (TransGen, HC301, dilution 1:3000) at room temperature for 1 h, and then probed with horseradish-peroxidase-conjugated secondary antibodies with a dilution of 1:10,000 (CellSignaling, 7076s) and developed with a chemiluminescent substrate (Millipore). Protein bands were visualized on the Tanon-6011C Chemiluminescent Imaging System (Tanon Science and Technology). All experiments were repeated at least four times.

For exogenous Co-IP assay, the GC cells were seeded on a 10 cm culture plate and co-transfected with different expression vectors. After 48 h, the cells were lysed with IP lysis buffer. About 1 mg of total protein was incubated with 5 µg of antibody against Flag (Sigma–Aldrich, F1804) or Myc-tag (TransGen Biotech, HT101e) or IgG overnight at 4 °C on a vertical roller. For endogenous IP assay, the 293 T cells were inoculated on a 10 cm culture plate and lysed with IP lysis buffer. The total protein was incubated with 5ug of antibody against FBXO31 or STYX or IgG. Then the mixture was incubated with 30 µL Protein A/G PLUS Agarose beads for 4 h. The immunoprecipitated proteins were identified by western blot.

Whole-cell extracts were prepared with 1% sodium dodecyl sulfate (SDS) lysis buffer with freshly added 1% proteinase inhibitor cocktail (MCE, HY-K0010) and 1mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, 32998-6). Protein concentrations were measured using the Pierce^TM BCA Protein Assay Kit (Thermo fisher, 23227) and equal amounts of proteins were loaded on SDS-PAGE gels. Immunoblotting analysis was performed as described previously 32 (link). The following primary antibodies were used with indicated dilution: Kindlin-2 (Proteintech, 11453-1-AP, 1:1000), DDX3X (Proteintech, 11115-1-AP, 1:1000), c-Myc (Cell signaling, 5605S, 1:1000), GAPDH (ABclonal, AC035, 1:5000), GFP (Transgen, HT801, 1:2000), Flag (Sigma, F1804, 1:3000), GST (Transgen, HT601, 1:2000) and MBP (Cell signaling, 2396S, 1:1000).