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Fig. S1. Myc-Sar1 GTPase detection in cell lines mycSar-Sec23HA and mycSarmut-Sec23HA. Representative images for Sec23HA and Sar1 GTPase deposition in cell lines mycSar-Sec23HA and mycSarmut-Sec23HA, using wide-field fluorescence microscopy. Sec23HA accumulation at ERES is not affected by overexpression of wild-type Sar1 GTPase (A) while it is clearly disrupted in the presence of mutated Sar1 GTPase (C). Both wild-type (B) and mutant (D) myc-tagged Sar1 GTPase show a diffused localization pattern. Nuclei were labelled with DAPI (blue). Insets: bright-field images. Scale bar: 1 μm. A SAG1 construct carrying a VSP trafficking signal and Sec23HA colocalize at ERES. (E) Representative image acquired by wide-field fluorescence microscopy of differentiating trophozoites expressing construct SAG1-VSPct. The SAG1 exodomain (red) was detected at the plasma membrane and at discrete intracellular sites reminiscent of ERES. Nuclei were labelled with DAPI (blue). Inset: bright-field image. Scale bar: 1 μm. (F) Representative image of a differentiating trophozoite coexpressing SAG1-VSPct (red) and Sec23HA (green) at ERES, acquired by wide-field fluorescence microscopy. Nuclei were labelled with DAPI (blue). Inset: bright-field image. Scale bar: 1 μm. (G) Confirmation of Gl-TPI-directed chromosomal integration of pPACV-Integ-derived constructs in cell lines Sec23HA, mycSec24-Sec23HA, mycSar-Sec23HA and mycSarmut-Sec23HA. Genomic DNA samples from untransfected cells (lane 1) and from cell lines Sec23HA (lane 2), mycSec24-Sec23HA (lane 3), mycSar-Sec23HA (lane 4) and mycSarmut-Sec23HA (lane 5) all present the control amplicons (c. 1100 bp) derived from a non-targeted locus. However, only samples derived from stably transfected lines (lanes 2–5) present the integration-derived c. 2000 bp amplicon, indicating that construct integration in gDNA of stably transfected lines occurred within the Gl-TPI locus. M, GeneRuler DNA ladder marker (Fermentas); selected band sizes in bp are indicated on the left.

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