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Blood, Vol. 113, Issue 1, 244-253, January 1, 2009
The Amot/Patj/Syx signaling complex spatially controls RhoA GTPase activity in migrating endothelial cells
Blood Ernkvist et al.
113: 244
Supplemental materials for: Ernkvist et al
Files in this Data Supplement:
- Table S1. Binding motif pull down (PDF, 29.7 KB)
- Figure S1. Amot family proteins bind to Syx (JPG, 24.8 KB)
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Peptides corresponding to the last 19 C-terminal amino acids of Amot, AmotL1 and AmotL2 including the PDZ-binding domains were used for pull-down experiments of mouse aortic endothelial lysate. Peptides lacking the three last amino acids were used as negative controls. Proteins that bound to peptides were separated by SDS PAGE and Syx binding to Amot, AmotL1 and AmotL2 was analyzed by western blot.
- Figure S2. Syx co-localizes with p80 Amot to lamellipodia (JGP, 113 KB)
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(A) Double immunofluorescent staining with antibodies against Syx and Amot of MAE cells retrofected with human p80 Amot, p130 Amot or empty vector as indicated in the figure. Syx staining overlaps with p80 Amot in lamellipodia of migratory cells. In contrast, in subconfluent cells, p130 Amot localizes to puncta along actin fibers. No overlap between Syx with p130 Amot could be detected. (B) Localization of Syx in bovine capillary endothelial cells expressing both isoforms of Amot. Here, Syx protein also localizes to lamellipodia but not to Amot staining in tight junctions. We have previously shown that p130 localizes to tight junction whereas p80 is detected in lamellipodia, vesicles and to a less extent in tight junctions.32
- Figure S3. Knockdown of syx-b in zebrafish (JGP, 95.3 KB)
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(A) Schematic representation of the relevant exons within syx-b. The syx-b MO1 morpholino was designed to target the 201 nt exon encoding the beginning of the Rho GEF domain. Primers for RTPCR amplification of wildtype and morphant splice products are shown as arrows below the exons. (B) RTPCR from syx-b MO1 and control injected embryos. Injection of syx-b MO1 results in an 86 nt insertion within the syx-b mRNA. (C) Sequencing of the RTPCR product obtained from syx-b MO1 injected embryos shows that the morpholino causes mis-splicing resulting and the inclusion of intronic sequence (red) within the syx-b morphant mRNA. Translation of this morphant RTPCR product shows the presence of a Stop codon within the intronic sequence resulting in premature truncation within the syx-b RhoGEF domain.
- Figure S4. Model of the protein complex that associates via the PDZ-binding motif of Amot (JPG, 30.3 KB)
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