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Blood, Vol. 110, Issue 2, 596-625, July 15, 2007
A novel negative regulatory function of the phosphoprotein associated with glycosphingolipid-enriched microdomains: blocking Ras activation
Blood Smida et al.
110: 596
Supplemental materials for: Smida et al, Vol 110, Issue 2, 596-605
Files in this Data Supplement:
- Figure S1. Anergic T cells showed no change in diacylglycerol kinase expression (JPG, 24.2 KB)
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One possible explanation for the block in Ras activation that would also bias signaling to favor calcium in anergic T cells would be an increased expression of diacylglycerol kinases (DGKs), the enzymes responsible for metabolizing diacylglycerol (DAG). However, probing of cell lysates from resting, anergic, and rescued T cells with anti-DGKα (left panel) or anti-DGKζ (right panel) revealed no change in DGK expression within these cells.
- Figure S2. Inhibition of Ras activation corresponds with PAG tyrosine phosphorylation (JPG, 53.3 KB)
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Jurkat T cells were transfected with vector, FPAG, FPAG-Y F mutants, FLAT, and Fyn, either alone or in the combination indicated. (A) corresponds to Figure 5A,B, (B) to Figure 5C, (C) to Figure 5D, and (D) to Figure 5E, respectively. Antibody staining shows expression of the corresponding construct. PAG phosphorylation is shown using pY317 staining. Note that due to overexpression, endogenous PAG staining is not observed. Actin staining shows equal loading.
- Figure S3. Transfection of Jurkat T cells with wild type FPAG enhances inhibitory tyrosine phosphorylation (JPG, 37 KB)
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Jurkat T cells were transfected with Fyn and either vector alone, wild type FPAG, or the Y317F mutant of FPAG that no longer recruits Csk. (A) Inhibitory tyrosine phosphorylation was analyzed to demonstrate that wild type FPAG indeed recruits more Csk to the membrane, as Y529 phosphorylation is an indicator of Csk activity. The blots were reprobed with actin to show equal loading and FLAG to demonstrate equal expression of both constructs. (B) Anti-FLAG immunoprecipitates were analyzed to show that although both constructs are tyrosine phosphorylated, the 317Y>F mutant no longer recruits Csk as efficiently as wild type FPAG. The blots were reprobed with anti-FLAG to show equal loading. (C) RasGAP immuno-precipitates from the same transfected cells as in panels A and B were probed with anti-FLAG to demonstrate that although mutation of Y317 disrupts Csk association, it does not affect RasGAP-PAG association. The blots were reprobed with anti-RasGAP to show equal loading.
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