HOP Regulates Cell Migration Figure 5. HOP colocalizes with microtubules and interacts with tubulin. A. 293T cells were transfected with GFP or GFP-HOP. Anti-GFP immunoprecipitation (IP) assay was performed to examine the interaction between HOP and tubulin. B. Purified tubulin was incubated with bacterially purified GST or GST-HOP immobilized on agarose. The presence of tubulin in the GST-pulldown preparation was examined by immunoblotting. C. Immunofluorescent images of HUVECs stained with anti-a-tubulin antibody, anti-HOP antibody, and DAPI to visualize microtubules (red), HOP (green), and nuclei (blue), respectively. D. A scheme summarizing the interactions of various forms of HOP with tubulin. E. 293T cells were transfected with GFP or various truncated forms of GFP-HOP. Cell lysates and anti-GFP immunoprecipitates were immunoblotted with anti-GFP and anti-a-tubulin antibodies. doi:10.1371/journal.pone.0036389.g005 the culture medium does not alter the ability of HUVECs to form tubes (Figure 6C and 6D). These results suggest that extracellular HOP might play a minor role, if there is any, in the migration of vascular endothelial cells. Discussion Angiogenesis, generation of new blood vessels from pre-existing ones, is required for many physiological or pathological processes. In embryo development, angiogenesis is important for providing nutrients and oxygen. During adulthood, angiogenesis occurs in response to some physiological stimuli [47,48], such as hypoxia and inflammation, for wound healing and repair. Angiogenesis also plays an important role in tumor growth, progression, and metastasis [24,25]. Thus, it is important to understand how the angiogenic process is precisely controlled. In the present study, by tube formation and capillary sprouting assays, we have identified HOP, a co-chaperone protein, as a novel regulator of angiogen- esis. In addition, we have investigated the underlying mechanism and found that HOP regulates angiogenesis by promoting endothelial cell migration. Several studies have shown that the microtubule cytoskeleton and associated proteins are involved in cell migration and polarization [44]. For example the deubiquitinase CYLD has been demonstrated to regulate angiogenesis by regulating PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e36389