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. Author manuscript; available in PMC: 2011 Dec 9.

Published in final edited form as: Mol Microbiol. 2010 Jun 9;77(4):912–929. doi: 10.1111/j.1365-2958.2010.07255.x

A Subcellular distribution of the SAG1-TM-CTD mutant chimeras by IFA and documented by confocal microscopy. Anti-GAP45 antibodies (in red), Anti-GRA3 (in red) and GRASP-YFP (in green) were used as IMC, DGs and Golgi markers, respectively. The nucleus was stained with DAPI (blue). pMS1tyMIC16m^TM-CTD, pMS1tyMIC8m^TM-CTD (in green) andpAS1tyAMA1m^TM-CTD (in red) expressing parasites were stained with anti-Ty-1. Scale bars indicate 5μm.

B–E Analysis of the cleavage events in the different chimeras. On the top, is shown the TMD of each TM-MIC in the original (wt) and mutated chimera (mut) and the residues mutated in the rhomboid cleavage motif are boxed. Below are western-blot analysis of lysates from parasites stably expressing the different pMS1tyMIC^TM-CTD fusion constructs. Indicated by arrows are the migration of the full (f) and shed (s) forms with indication of the molecular weight. B Migration of pMS1tyMIC16^TM-CTD (wt) and pMS1tyMIC16m^TM-CTD (mut). C Migration of pMS1tyMIC8^TM-CTD (wt) and pMS1tyMIC8m^TM-CTD (mut). D Migration of pMS1tyMIC8.2^TM-CTD. E Migration of pAS1tyAMA1^TM-CTD. The proteins were detected with anti-Ty-1. Molecular weight markers are indicated in kDa.

A Subcellular distribution of the SAG1-TM-CTD mutant chimeras by IFA and documented by confocal microscopy. Anti-GAP45 antibodies (in red), Anti-GRA3 (in red) and GRASP-YFP (in green) were used as IMC, DGs and Golgi markers, respectively. The nucleus was stained with DAPI (blue). pMS1tyMIC16m^TM-CTD, pMS1tyMIC8m^TM-CTD (in green) andpAS1tyAMA1m^TM-CTD (in red) expressing parasites were stained with anti-Ty-1. Scale bars indicate 5μm.

B–E Analysis of the cleavage events in the different chimeras. On the top, is shown the TMD of each TM-MIC in the original (wt) and mutated chimera (mut) and the residues mutated in the rhomboid cleavage motif are boxed. Below are western-blot analysis of lysates from parasites stably expressing the different pMS1tyMIC^TM-CTD fusion constructs. Indicated by arrows are the migration of the full (f) and shed (s) forms with indication of the molecular weight. B Migration of pMS1tyMIC16^TM-CTD (wt) and pMS1tyMIC16m^TM-CTD (mut). C Migration of pMS1tyMIC8^TM-CTD (wt) and pMS1tyMIC8m^TM-CTD (mut). D Migration of pMS1tyMIC8.2^TM-CTD. E Migration of pAS1tyAMA1^TM-CTD. The proteins were detected with anti-Ty-1. Molecular weight markers are indicated in kDa.