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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Traffic. 2010 Feb 15;11(5):587–600. doi: 10.1111/j.1600-0854.2010.01050.x

Figure 1.

Figure 1

Dynamics of Sec22b, calnexin and Rab1b recruitment to vacuoles containing L. pneumophila after silencing of plasma membrane syntaxins. A) Cell lysates were obtained from HEK293-FcγRII cells transfected for 72 h with the siRNA indicated (top), and immunoblot analysis was used to measure the levels of the proteins indicated (left). Calnexin levels were used to assess equal protein loading. B) HEK293-FcγRII cells were treated with the indicated siRNA or mock treated as indicated in the upper left corner of each image panel. Cells were fixed and stained with antibodies specific for the proteins indicated on the left of each row. FcγRII staining was conducted to determine if the triple syntaxin knockdown (Stx2/3/4 KD) affected the localization of this plasma membrane protein. Scale bar = 5 μm. C and D) HEK293-FcγRII cells were transfected with siRNA targeting the indicated syntaxins (Stx2, Stx3, Stx4, Stx2/3/4) or treated with control siRNA (mock) as indicated at the bottom of the graph. Cells were fixed either 1 (C) or 4 h (D) after infection with wild-type L. pneumophila, and stained with antibodies to either Sec22b, calnexin or Rab1b as indicated in each panel. Colocalization of Sec22b, calnexin and Rab1b was assessed by fluorescence microscopy and the data are presented as the percent of vacuoles that were positive for each marker. Data are the mean ± SEM of three independent experiments in which 100 vacuoles were scored [*p < 0.02 and **p < 0.04 (C), *p < 0.002 and **p < 0.001 (D), as compared with mock-treated]. E) HEK293-FcγRII cells were transfected with an siRNA pool to silence the production of the plasma membrane-localized syntaxins (Stx2/3/4; black bars) or treated with control siRNA (mock; white bars) as indicated. Cells were fixed 8 h after infection with wild-type L. pneumophila. Intracellular replication of L. pneumophila was assessed by counting the number of bacteria residing in a single vacuole in the infected cells. The data are presented as the percent of vacuoles in mock-transfected cells (white bars) and Stx2/3/4 siRNA-transfected cells (black bars) that contained 1–3 bacteria, 4–6 bacteria or >7 bacteria. Data are the mean ± SEM of three independent experiments in which 100–120 vacuoles were scored (*p < 0.009 and **p < 0.02, as compared with mock-treated).