Fig. 1.

Phosphoproteome analysis of cells infected with F. novicida. A, Experimental design. RAW 264.7 macrophage-like cells were infected for 30 min with wild-type (WT) Francisella or with ΔlpcC, followed by incubation for 0–4 h before harvesting. A negative control was run in parallel using media alone (Mock). After infection, cells were harvested, digested with trypsin and the resulting peptides were labeled with iTRAQ. Equal parts of each iTRAQ sample were combined and phosphopeptides were captured by immobilized metal affinity chromatography (IMAC). Enriched phosphopeptide fractions were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) leading to the identification of 3008 phosphopeptides. B, Differentially abundant phosphopeptides from RAW 264.7 cells after Francisella infection. Abbreviations: L, ΔlpcC-infected; M, mock-infected; n, number of phosphopeptides; W, wild type-infected.