Issues associated with the use of phosphospecific antibodies to localise active and inactive pools of GSK-3 in cells

<p>Abstract</p> <p>Background</p> <p>Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine (Ser/Thr) kinase comprising two isoforms, GSK-3α and GSK-3β. Both enzymes are similarly inactivated by serine phosphorylation (GSK-3α at Ser21 and GSK-3β at Ser9) and activated by tyrosine phosphorylation (GSK-3α at Tyr279 and GSK-3β at Tyr216). Antibodies raised to phosphopeptides containing the sequences around these phosphorylation sites are frequently used to provide an indication of the activation state of GSK-3 in cell and tissue extracts. These antibodies have further been used to determine the subcellular localisation of active and inactive forms of GSK-3, and the results of those studies support roles for GSK-3 phosphorylation in diverse cellular processes. However, the specificity of these antibodies in immunocytochemistry has not been addressed in any detail.</p> <p>Results</p> <p>Taking advantage of gene silencing technology, we examined the specificity of several commercially available anti-phosphorylated GSK-3 antibodies. We show that antibodies raised to peptides containing the phosphorylated Ser21/9 epitope crossreact with unidentified antigens that are highly expressed by mitotic cells and that mainly localise to spindle poles. In addition, two antibodies raised to peptides containing the phosphorylated Tyr279/216 epitope recognise an unidentified protein at focal contacts, and a third antibody recognises a protein found in Ki-67-positive cell nuclei. While the phosphorylated Ser9/21 GSK-3 antibodies also recognise other proteins whose levels increase in mitotic cells in western blots, the phosphorylated Tyr279/216 antibodies appear to be specific in western blotting. However, we cannot rule out the posssibility that they recognise very large or very small proteins that might not be detected using a standard western blotting approach.</p> <p>Conclusions</p> <p>Our findings indicate that care should be taken when examining the subcellular localisation of active or inactive GSK-3 and, furthermore, suggest that the role of GSK-3 phosphorylation in some cellular processes be reassessed.</p> <p>Reviewers</p> <p>Dr. David Kaplan, Dr. Robert Murphy and Dr. Cara Gottardi (nominated by Dr Avinash Bhandoola.)</p

Smarte Unternehmer, ausgegrenzte Versager: produktives Scheitern im 15. Jahrhundert?

In late medieval economy, failing was as much a social as an economic process. The story of the hanseatic merchant Hildebrand Veckinchusen (ca. 1365-1426) shows how his network of family members, friends, and business partners on the one hand enabled him to be successful as a merchant. On the other hand the same network deployed its social power against him once he had run into problems: As his potential new investors had to stem from the circle of his old partners, they all knew his fate, and they all informed each other about his shortcomings. So Hildebrand had to borrow the money he needed from outside his network from an external creditor. His friends would have been able to defer the return of his loans (as they knew how to keep track of him), whereas the external creditor could not and thus put him in prison as an insolvent debtor. His friends offered help only on the condition that Hildebrand did admit his faults and listened to their advice, but he refused. But Veckinchusen’s network demanded control of his decisions and affairs in which the social an economic sphere were intimately linked. Hildebrand didn’t perceive himself as failed, contrary to what his friends expected him to do. Thus he wasn’t allowed back into the network, which, to be successful, had to exclude failures such as Hildebrand Veckinchusen

Automatic Air Traffic Control Part I: Projected System

Coordinated Science Laboratory changed its name from Control Systems LaboratoryContract DA-36-039-SC-8512