-Triton and incubated with goat anti-mouse Alexa Fluor 555 diluted 1: 100 in 1 PBS for visualisation and counterstained for the nucleus with Hoescht. Images were taken at 40 objective using a microscope. CD31 Immunohistochemistry. Serial 7-mm sections of PFA-fixed paraffin wax-embedded tumour parts were mounted onto slides. Vessel presence was confirmed by CD31 staining. Sections were blocked in 5% normal goat serum for 30 min, CD31 antibody was added overnight, 2 mg ml 1 anti-rabbit biotin antibody for 1 h followed by the avidinbiotinylated enzyme complex for 30 min followed by the DAB substrate. Sections were co-stained with haematoxylin and examined using a Nikon Eclipse E400 microscope, and photos were captured using Nikon Eclipse Net software. In vivo tumour model. All animal experiments were carried out under a UK Home Office License after approval by the University of Bristol Ethical Review Group. A375, A375 shRNA control and A375 shRNA SRPK1 knockdown cells were cultured in T75 flasks to 80% confluence. Trypsinised cells were counted using a haemocytometer, and 2 million cells of A375 shRNA control and A375 shRNA SRPK1 were injected subcutaneously either into the left and right flanks of nude mice, or a single injection of untransduced A375 cells. Tumour-bearing mice were weighed and tumours were measured by caliper bi-weekly. Mice bearing A375-untransfected tumours were treated with either 100 ml of 20 mg ml 1 SRPIN340, or 100 ml of 1% DMSO vehicle control injected daily into the peritumoral space. Tumour volumes were calculated according to the formula /2) where A length of the tumour and B tumour width. The mice were culled by cervical dislocation Act 1986), when the first PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19809023 tumour in each experimental groups reached 16 mm maximum length. The tumours were excised, weighed and either stored in 4% PFA diluted in 1 PBS or frozen down to 80 1C for further analysis. Investigators were blinded to each group when performing injections, taking measurements and analysing data. Statistical analysis. Data are shown as means.e.m. All data, graphs and statistical analyses were calculated with Microsoft Excel, GraphPad Prism and ImageJ. All results were considered statistically significant at Po0.05, Po0.01 and Po0.001. RESULTS SRPK1 acts through SRSF1 to regulate pro-angiogenic VEGF expression in melanoma cells. To determine the expression of SRPK1 in different melanoma cell lines, mRNA extracted from CM and UM cell lines was assayed by qRTPCR and compared with YM-155 primary pigmented epithelial cells. All melanoma cell lines expressed significantly more SRPK1 than primary RPE cells with A375 expressing significantly more than UM cell lines, which expressed between 20 and 50% of the levels found in A375 cells relative to internal controls. At the protein level, SRPK1 and SRSF1 was expressed in all melanoma cell lines with higher expression levels of SRPK1, and possibly SRSF1 appearing in metastatic cell lines, A375 and Omm2.5 than in Mel270 and 92.1. A similar relative expression of VEGF165 expression at the RNA level was seen and total VEGF was higher in A375, Omm2.5 and Mel270 than 
92.1, whereas the anti-angiogenic VEGFxxxb isoforms appeared stronger in the primary compared with metastatic cell lines., suggesting that the pro-angiogenic isoforms were raised in metastatic cell lines relative to Mel270 and 92.1. SRPK1 has been inextricably linked to the phosphorylation of the splicing factor SRSF1. Inhibition of SRPK1 by SRPIN340 prevents the nuc