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. 2013 Dec;33(24):4909–4918. doi: 10.1128/MCB.00565-13

Fig 3.

Fig 3

Localization and GEF activity of Syx are required for Syx effects on cell polarity and migration. (A) Schematic domain structures of murine pEYFP-Syx, pEYFP-Syx-ΔPBM, pEYFP-Syx-N596A, and pEYFP-Syx-ΔDH. DH, Dbl homology; PH, pleckstrin homology; PDZ-BM, PDZ-binding motif. (B) Immunofluorescence experiment comparing the morphology of Syx-depleted U251 cells expressing pEYFP-Syx, pEYFP-Syx-ΔPBM, or pEYFP-Syx-N596A. The framed regions are shown separately to highlight the localization of expressed constructs; arrows indicate pEYFP-Syx or pEYFP-Syx-N596A that localized at cell edges. Fixed cells were stained for YFP and α-tubulin. (C) Western blot analysis of active and total RhoA from lysates of HeLa cells expressing the indicated v5-tagged constructs. (D) Transwell migration of control shRNA versus syx shRNA expressing HeLa cells toward the FBS chemoattractant. Migration data are expressed as the % control (mean ± the SD; n = 9). Western blot analysis shows the knockdown efficiency of the shRNAs. (E) Transwell migration of HeLa cells transiently expressing pEYFP, pEYFP-Syx, pEYFP-Syx-ΔPBM, or pEYFP-Syx-ΔDH. Migration data are expressed as the % control (mean ± the SD; n = 9). Western blot analysis of active RhoA in lysates of HeLa cells expressing the indicated constructs.