Fig. 2: Flavivirus infection triggers activation of three specific SFKs.

a Large-scale immunoprecipitation of activated SFKs was performed on anti-phospho-SFK antibodies from mock- and Dengue-infected BHK21 and Vero E6 cells (MOI 2, 24 h). Isolated proteins were resolved by SDS-PAGE and detected by silver staining. b Entire lanes on gels were sliced into 2-mm sections and subjected to trypsin digest. The peptide mix was processed and analysed by an LTQ Orbitrap mass spectrometer. c Protein expressions of Lyn, Fyn and Src kinases were validated in lysates prepared from indicated Dengue-susceptible cell types. d Activation of Lyn, Fyn and Src upon Dengue infection was measured by immunoprecipitating first on anti-phospho-tyrosine antibodies and immunoblotting with specific SFK antibodies. e Reciprocal immunoprecipitation on specific anti-SFK antibodies followed by immunoblotting with anti-phospho-tyrosine antibodies. f Activation of SFKs was measured in lysates prepared from Dengue-infected cells with the Milliplex MAP 8-plex assay kit using the Luminex system as read-out, following the manufacturer’s protocol. Red bars indicate Lyn activation. Error bars represent mean ± s.d. from three biological replicates. g Colocalisation of Dengue and SFKs was visualised by 4G2 antibodies and anti-phospho-SFKs with markers for ER (Pdi), Golgi (Golgin97) and autophagosomes (LC3) as controls. Insets indicate colocalisation of markers with pSFKs. Images are representative of five independent experiments. Scale bars represent 20 µm. h Manders correlation coefficients were measured for colocalisation of pSFKs with E protein, Golgi, ER and autophagosome marker proteins from 50 cells per condition (error bars represent mean ± s.e.m. of five independent experiments). i, j Same as g, h in Zika-infected cells (error bars represent mean ± s.e.m. of five independent experiments).