5 protocols
Array processing were performed as recommended by the array manufacturer (Affymetrix).
Hybridization, and array processing were performed as recommended by the array manufacturer (Affymetrix). Each ChIP assay was performed in biological triplicate and hybridized to GeneChip Human Tiling 2.0R Array Set (Affymetrix).
Terminal labeling was performed as recommended by the array manufacturer (Affymetrix).
ChIP-chip was performed essentially as described previously (O’Geen et al. 2006. Biotechniques 41:577–580) with the following modifications: LECs were cross-linked with formaldehyde in complete endothelial cell basal medium MV (PromoCell), and IP dilution buffer was substituted with 3% BSA containing complete inhibitor tablets (Roche). DNA was sheared using a sonicator (Soniprep 150; MSE) and immunoprecipitated using mouse anti-Myc antibody (clone 9E10, Santa Cruz Biotechnology). Decrosslinked ChIP DNA was amplified using GenomePlex Complete Whole Genome Amplification kit (Sigma-Aldrich) using the buffer supplemented with deoxynucleotide-triphosphate + deoxy-UTP mixture. DNA was fragmented using uracil DNA glycosylase and apurinic/apyrimidinic endonuclease.
Primary human lymphatic endothelial cells (LECs) were isolated and cultured as described previously (Norrmen et al. 2010. Blood 115(4): 906-909). Lymphatic endothelial identity was confirmed by lymphatic marker PROX1 staining. LECs were transduced with 20 plaque-forming units/cell of recombinant adenoviruses expressing Myc-tagged human transcription factor FOXC2 (Petrova et al. 2004. Nat Med 10(9): 974-981) or its phosphorylation-deficient mutant (Myc-pmFOXC2) in which eight phosphorylation sites (S219, S232, S240, T247, S251, S281, S288 and S367) were replaced by alanine.