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. 2018 Dec 3;217(12):4199–4214. doi: 10.1083/jcb.201802155

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© 2018 Singh et al.

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Figure 4. — Salmonella:sipA^{431FLAG-685HA} recruits Syn8 on SCVs. (A) Top: Schematic diagram showing the position of FLAG and HA tag in the sipA^{431FLAG-685HA} construct. Bottom: Western blot analysis showing the secretion of SipA^{431FLAG-685HA} like SipA:WT protein in the culture supernatant (SSPs). (B) HeLa cells were infected with Salmonella:sipA^{431FLAG-685HA}, and localization of SipA 90 min p.i. was determined using anti-FLAG or anti-HA antibody in respective cells (green). Salmonella was stained with anti-Salmonella antibody (red). (C) Similarly, cells were infected with Salmonella:sipA^{431FLAG-685HA} for indicated times, and localization of SipA in the same cells was detected using anti-FLAG (green) and anti-HA (red) antibodies to determine intracellular cleavage of SipA. (D) To determine the colocalization of SipA with Syn8, cells were infected with Salmonella:sipA^{431FLAG-685HA} and stained with anti-FLAG or anti-HA antibody 90 min p.i. Cells were also costained with phalloidin (actin) or anti-Syn8 antibody as indicated. Arrows indicate colocalization of the respective proteins (C) or on SCVs (B and D). All results are mean ± SEM of three independent experiments, and levels of significance are indicated by P values below the respective figures.