As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

pmic7453-sup-0001-figureS1.jpg36KFigure S1 - Analysis of the specificity of binding of biotin-15d-PGJ2 and target validation. (A) ECs lysates were pre-incubated with 10 or 100μM of unlabelled 15d-PGJ2 and then treated with 5μM biotin-PGJ2 Proteins were affinity precipitated using Neutravidin beads and separated by 1D electrophoresis, followed by anti-biotin Western blot. These data suggest that biotin-tagged 15d-PGJ2 and unlabelled 15d-PGJ2 target the same sites in proteins, as can be seen from the pre-treatment of the lysate with molar excess of non-labelled 15d-PGJ2. 5μM of unlabelled 15dPGJ2 was used as negative control (B) To further assess the specificity of the assay, the previous membrane was also stained to detect one of the 15d-PGJ2 target protein identified in the pull-down, exportin-1. 5μM of unlabelled 15d-PGJ2 was used as negative control. (C) The validation of exportin-1 as a target of 15d-PGJ2 has been carried out by immunoprecipitating exportin-1 protein from EC treated with 5μM biotin-15d-PGJ2 and detecting the modification by anti-biotin WB. 5μM of unlabelled 15d-PGJ2 was used as negative control. Total cell lysate was used as positive control (pos. control). All experiments were carried out independently three times.
pmic7453-sup-0002-TableS1.doc487KTable S1. List and details of 15d-PGJ2 candidate target proteins. Candidate target proteins identified by mass spectrometry following biotin-15d-PGJ2 affinity purification and subtraction of proteins identified in control experiments where the biotin-15d-PGJ2 was replaced by unlabelled 15d-PGJ2 or biotin-PGD2. In total, 422 proteins were identified before subtraction of the proteins identified in the control experiments. All lists have been submitted to PRIDE. Proteins validated by additional immumoaffinity and Western blot are highlighted. In each case, Western blot using an antibody to biotin confirmed the modification of the protein. Two proteosomal proteins not seen in the biotin-15d-PGJ2 candidate target proteins were similarly analysed and shown not to be modified by biotin Western blot.
pmic7453-sup-0003-suppmat.doc30Ksupplementary material

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.