Chikungunya virus phosphoproteomics

SILAC-labeled MRC-5 cells were seeded in 75-cm2 flasks 1 day before infection with CHIKV-LS3 [1] at MOI 5. One hour post infection (h p.i.), the inoculum was removed and replaced with SILAC DMEM containing 2% dialyzed FBS, 0.280 mM arginine, 0.384 mM lysine, 0.5 mM proline, 25 mM HEPES, 2 mM L-Glutamine and 1% NEAA. At 2, 8, and 12 h p.i., infected and mock-infected cells were harvested for phosphoproteomics analysis by lysis in 4% SDS, 0.1M Tris pH 7.6, followed by heating to 96°C for 10 min. At 12 h p.i,. protein lysates for western blot (WB) analysis were harvested in 4× Laemmli sample buffer (LSB) (100 mM Tris-HCl, pH 6.8, 40% glycerol, 8% SDS, 40 mM DTT, 0,04 mg/ml bromophenol blue) and cells grown on coverslips were fixed in 3% PFA in PBS . The experiment was performed in duplicate with a label swap.