Effects of MCFA on cardiac function were previously reviewed by Francois Labarthe and colleagues

ol reading at 570 nm. Revelation Quicklink 4.25 software was also used to calculate a standard curve from mean absorbance values of standards enabling the estimation of VEGF concentration for each sample. Western blotting. Protein samples were mixed with 1 SDS loading buffer bromophenol blue and 5% final concentration 2-mercaptoethanol, pH 6.8). To denature the protein, samples were boiled for 5 min at 100 1C. Samples were subjected to polyacrylamide gel electrophoresis on a 12% SDSPAGE gel at 90 V in ice cold running buffer for B2.5 h. The separated proteins were then electrophoretically blotted to a methanol-activated polyvinylidene fluoride membrane by wet transfer for 2 h at 90 V in transfer buffer. Membranes were incubated in blocking solution Cell culture and transfection. CM cell line, A375, and UM cell lines, 92.1, Mel270 and Omm2.5 cells were cultured in DMEM, 10% FBS, 0.5% PenStrep and split at 80% confluence. A375 melanoma cells were treated with culture medium plus 6 mg polybrene and 1 ml scrambled shRNA or 5 ml SRPK1 shRNA per 200 000 cells. Cells transduced with the lentivirus were selected using puromycin at 2 mg ml 1. Transduction efficiency was determined by green fluorescent protein expression. Pharmacological inhibitor treatments. SRPIN340 -5-phenyl] isonicotinamide), was purchased from Ascent Scientific, Bristol, UK. Cells at B70% confluence were serum starved for at least 12 h and treated with 110 mM SRPIN340, 0.020.05% DMSO was added to vehicle control. After 24 h, mRNA was extracted or cells were fixed for staining, 48 h later protein was extracted, for further analysis. Cell viability assays. Cell proliferation was determined by two methods. Thirty thousand A375 cells per well, transduced with scrambled shRNA, SRPK1 shRNA or untransduced and treated with SRPIN340, were seeded on 24-well plates. Every 24 h cells were trypsinised and a cell count was performed. Cells seeded on cover slips were also stained for Ki67. For scratch assays, cells were grown to confluence in 24-well plates and a 1-mm-thick line of cells was 478 SRPK1 inhibition is a novel targeted therapy BRITISH JOURNAL OF CANCER with agitation at room temperature for 30 min and then probed with the primary antibody overnight at 4 1C; rabbit polyclonal anti-VEGF-A diluted 1: TG-101348 web 1000-1: 100 in 2.5% non-fat dried milk TBS-T, VEGFxxxb-specific mouse monoclonal 56/1 diluted 1: 1000-1: 500 in 5% BSA TBS-T, media from mouse hybridoma cell line, mab104 diluted 1: 4 in TBST and goat polyclonal beta-tubulin diluted 1: 1000 in 5% BSA TBS-T. Membranes were then washed four times for 10 min each with TBS-0.3%T before incubation with secondary HRP-conjugated antibodies: goatamouse immunoglobulin G, goatarabbit IgG or rabbitagoat IgG diluted 1: 10 000 in 5% BSA PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19808515 TBS-T or 2.5% non-fat dried milk TBST, for 45 min at room temperature with agitation. The washes were repeated and the bands were detected using the Enhanced Chemoluminiscence SuperSignal West Femto Maximum Sensitivity Substrate kit. Immunofluorescence staining and imaging. For immunofluorescence, cells were grown to 80% confluence on glass cover slips. After treatment the cells were washed with PBS, fixed for 5 min with 4% PFA and then washed with PBS in 0.05% Triton X. The cells were blocked in 5% normal goat serum in PBS-TritonX for 1 h, washed thrice with PBS-TritonX and incubated overnight with 2 mg ml 1 of mouse monoclonal SRSF1 or 2 mg ml 1 mouse monoclonal anti-SRPK1. The cells were washed thrice with PBS