Fig. 3: VgrG4 colocalizes with the ER. | Nature Communications

Fig. 3: VgrG4 colocalizes with the ER.

From: A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection

Fig. 3

VgrG4 localisation within the cells was analysed by confocal microscopy of A549 mCherryER cells (ER in red). Cells were infected with YeVgrG4 for 90 min, or Kp52145 for 3 h, or left uninfected (n.i.) VgrG4 was stained with VSV-G or FLAG (in green) (a). b Immunoblot analysis of VgrG4-tagged with VSV-G, Calnexin (ER marker) and Tom20 (mitochondria marker) in fractionated lysates of A549 cells infected with YeVgrG4 for 90 min or left uninfected (n.i.). c Confocal microscopy of A549 mCherryER cells infected with Y. enterocolitica encoding N- and C-terminal truncated forms of VgrG4 tagged with VSV-G for 90 min. To determine the tethering protein associating with VgrG4, A549 mCherryER cells were transfected with siRNAs for VAPB or MFN2 and infected with YeVgrG4 for 90 min (d). e Confocal microscopy of A549 cells transfected with the MFN2-YFP plasmid (MFN2 in green) and infected with YeVgrG4 for 90 min. VgrG4 was stained with VSV-G. f Immunoblot analysis of VSV-G (VgrG4) and GFP (MFN2) levels in immunoprecipitates of A549. 24 h after transfection with a MFN2-YFP plasmid, cells were infected with YeVgrG4 for 90 min. Lysates were immunoprecipitated using anti-GFP antibody, and membranes were first probed with antibody against VSV-G and subsequently with antibody against GFP. Pre-immune mouse IgG served as negative control. All images and immunoblots are representative of three independent experiments.

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