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Blood, Vol. 109, Issue 2, 552-559, January 15, 2007
IRAG mediates NO/cGMP-dependent inhibition of platelet aggregation and thrombus formation
Blood Antl et al.
109: 552
Supplemental materials for: Antl et al, Vol 109, Issue 2, 552-559
Methods In-gel digestion, sample preparation and mass spectrometry. Coomassie brilliant blue stained protein bands were excised, washed, reduced, alkylated and digested with trypsin, followed by extraction and desalting of the peptide mixture as previously described.1 For nanoelectrospray-based precursor ion scanning2 the peptides were stepwise eluted with 50% methanol and then 50% methanol/5% ammonia from the desalting column into the spraying needle. For peptide sequencing1 in positive ion mode the peptides from an aliquot of the sample were eluted with 50% methanol/5% formic acid. Detection of phosphopeptides was performed as previously described2 using precursor ion scanning for the phosphate-derived anion m/z -79 with a nanoelectrospray source mounted on a triple quadrupole instrument (API III,PE-Sciex, Ontario Canada). Generation of phosphospecific IRAG antibodies. Phosphopeptides containing Ser664 (CARSMpSLTLGK) corresponding to amino acids 659-668 or Ser677 (CRRVpSVAV) corresponding to amino acids 674-680 of human IRAG were synthesized and purified by HPLC (Covalab, France; Gramsch Laboratories, Germany). Then, the peptides were conjugated to KLH by MBS and injected into rabbits. IgG were purified from the obtained antisera using Protein A (Amersham) and Sulfolink coupled peptide (Pierce). Phosphorylation in COS-7 cells. COS-7 cells transiently transfected with bovine wt IRAG or mutants cloned in pcDNA3.1-vector and cGKI in pMT3-vector were harvested and lysates of these cells were phosphorylated in the presence or absence of 8-pCPT-cGMP (3 µM) (2 min, 30°C) as described.3 Proteins were analyzed by SDS-PAGE and Western blot followed by immunodecoration or autoradiography. References 1. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996;68:850-858
2. Neubauer G, Mann M. Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: potentials and limitations. Anal Chem. 1999;71:235-242
3. Ammendola A, Geiselhoringer A, Hofmann F, Schlossmann J. Molecular determinants of the interaction between the inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) and cGMP kinase Ibeta. J Biol Chem. 2001;276:24153-24159
Files in this Data Supplement:
- Figure S1. cGKI macrocomplex in murine wild-type and IRAGΔ12/Δ12 platelets (PDF, 149 KB) -
(A) Detection of IRAG, cGKI , cGKI, and IP3RI in platelets from wild-type (lane 1), heterozygous IRAG+/Δ12 (lane 2), and IRAGΔ12/Δ12 mice (lane 3). Amount of loaded protein was 70 µg as control protein MAPK was used. (B) Analysis of VASP phosphorylation in wild-type (WT) and IRAGΔ12/Δ12 ( 12) platelets. Phosphorylation of VASP in intact platelets was stimulated by DEA/NO (10 µM, 1 or 5 minutes) or 8-pCPT-cGMP (100 µM, 10-30 minutes) and analyzed by pSer239-VASP antibody. Control conditions were performed for DEA/NO adding a final concentration of 1 mM NaOH and for 8-pCPT-cGMP adding H2O. (C) Isolation of the cGKI macrocomplex from wild-type (lane 1), IRAG+/Δ12 (lane 3), and IRAGΔ12/Δ12 (lane 5) platelets. Using cGMP affinity chromatography, the complex from wild-type (lane 2), IRAG+/Δ12 (lane 4), and IRAGΔ12/Δ12 (lane 6) platelets was purified and analyzed with IRAG-, cGKI-, and IP3RI-specific antibodies.
- Figure S2. Specificity of pSer677-IRAG and pSer664-IRAG antibodies (PDF, 65.4 KB) -
(A-B) 32P-phosphorylation with (+) or without (−) 8-pCPT-cGMP (3 µM) in lysates from COS-7 cells transfected with cGKI and bovine IRAG wt (IRAG) or its Ser696/Ala variant (IRAGRRVS/A), corresponding to Ser677/Ala mutation of human IRAG (A), or its Ser683/Ala variant (IRAGRSMS/A), corresponding to Ser664/Ala mutation of human IRAG (B), analyzed by autoradiography (AR) and immunoblot (IB) using pSer677-IRAG antibody (pSer677-IRAG-Ab) (A) or pSer664-IRAG antibody (pSer664-IRAG-Ab) (B). Equal abundance of total IRAG in the cells was analyzed by immunoblotting with the IRAG antibody (IRAG-Ab). (C) Summary of experiments shown in Figure 2A. The stimulation of Ser664-IRAG phosphorylation after incubation of human platelets with 8-pCPT-cGMP (cGMP) or DEA/NO was calculated in comparison to the basal phosphorylation of Ser664 in the control reactions.
- Figure S3. Effect of cBIMPS and prostacyclin on collagen-induced aggregation of wild-type and IRAGΔ12/Δ12 platelets (PDF, 51.4 KB) -
Statistical evaluation of platelet aggregation (top) and shape change (bottom) from the experiments shown in Figure 3A.
- Video 1. WT + vehicle (MOV, 4.62 MB)
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Videos 1-4 show thrombus formation in the injured carotid artery of WT or IRAGΔ12/Δ12 mice in the presence or absence of nitric oxide. These videos are provided for illustration.
- Video 2. WT + NO (MOV, 3.62 MB)
- Video 3. IRAGΔ12/Δ12 + vehicle (MOV, 4.24 MB)
- Video 4. IRAGΔ12/Δ12 + NO (MOV, 3.06 MB)
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Bars indicate 50 µm.
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