Protein g sepharose beads

For the co-immunoprecipitation experiments, SH-SY5Y cells were washed with ice-cold PBS and harvested in 1% (v/v) Triton X-100 (in PBS) 48 h after transfection. The lysates were rotated at 4 °C for 1 h and then centrifuged at 22,250× g for 20 min. The supernatants were combined with protein G Sepharose beads (Amersham Biosciences, Piscataway, NJ, USA), pre-incubated with the antibody against Myc (A190-105A, Bethyl Laboratories, Montgomery, TX, USA), and mixed by rotation overnight at 4 °C. The following day, protein G Sepharose beads (Amersham Biosciences, Piscataway, NJ, USA) were pelleted and washed thrice with 1% (v/v) Triton X-100. The precipitates were resolved using SDS-PAGE and subjected to immunoblot analysis.

Cell lysates (CL) were pre-cleared by incubation with 30 µl of 50% Protein G Sepharose beads (Amersham Pharmacia Biotech) for 1 h. Pre-cleared lysates from mock- or DENV-infected cells were then incubated for overnight at 4 °C with 30 µl of 50% Protein G Sepharose beads conjugated to pSFKs (for phosphorylated SFK screen) or P-Tyr-100-conjugated magnetic beads (to validate selected SFK candidates). Subsequently, beads were collected by centrifugation at 13,000 rpm for 30 s at 4 °C and washed three times with cold wash buffer (50 mM Tris-HCl buffer, pH 7.4, containing 0.1% (wt/vol) Triton X-100, 300 mM NaCl and 5 mM EDTA) supplemented with 0.02% (wt/vol) sodium azide and phosphatase inhibitor tablets, and once with cold PBS. Bound proteins were eluted by boiling in 30 µl of 2×SDS-PAGE loading buffer, separated by gel electrophoresis and visible in silver stain or analysed by western blotting using appropriate antibodies.

Cell lysates were prepared with 0.5 ml of RIPA buffer per 100 mm culture dish. Cell lysates were diluted in NET buffer (50 mmol/LTris-HCl, pH 7.4, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.05% Nonidet P-40) and incubated overnight at 4°C with 2 μg of anti-XB130, anti-phosphotyrosine, or anti-cortactin antibody. Three μg of protein G-sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA) were added to the cell lysate-antibody mix and incubated at 4°C for 1 h. Bead-bound complexes were washed three times with cold NET buffer and denatured in Laemmli buffer at 100°C for 5 min. Samples were analysed by western blotting.

Immunoprecipitation of protein complexes was performed as described previously [30 (link)], with minor modifications. Cells were lysed in buffer L (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 10 mM NaF, Roche Complete protease inhibitor cocktail), incubated on ice for 30 min and sonicated with a Bioruptor Plus (Diagenode, Denville, NJ, USA) for 10 cycles (30 s on, 30 s off) at 4 °C with power set to high. Lysates were pre-cleared by centrifugation at 15,000 rpm for 15 min and immunoprecipitated using either mouse anti-Flag M2 monoclonal antibody (1:400 dilution; Sigma #F3165), goat anti-Flag (OctA-Probe D-8) polyclonal antibody (1:30 dilution; Santa Cruz Biotechnology sc-807-G), rabbit anti-TRIB3 polyclonal antibody (1:300 dilution; Calbiochem #ST1032, Burlington, MA, USA) or, as a negative control antibody, mouse anti-E2Tag monoclonal antibody (1:120 dilution; Quattromed) with rotation overnight at 4 °C. The immunocomplexes were collected from the lysate with Protein G sepharose beads (Amersham Biosciences) and eluted in SDS gel sample buffer. The eluted samples were resolved on 10% SDS-PAGE and analyzed by Western blotting as described above.