Imagequant las 4000 biomolecular imager software

Western blot analysis was performed using an Epithelial-Mesenchymal Transition Antibody Sampler kit (N-cadherin, E-cadherin, Vimentin, Claudin-1, β-catenin, Snail, Slug and TCF8/ZEB1 antibodies), Integrin Antibody Sampler Kit (Integrin α4, α5, αV, β1, β3, β4 and β5 antibodies), anti-FAK, anti-PI3K, anti-AKT, anti-p-AKT (Ser473), anti-ERK1/2, anti-p-ERK1/2, anti-Notch1, anti-EGFR (Cell Signaling Technology), anti-Twist, anti-fibronectin (FN) (Abcam), anti-FOXM1, anti-Arp2, anti-Arp3, anti-AMFR (GeneTex), and anti-GAPDH antibodies (Millipore). The chemiluminescent signals were visualized and quantified using ImageQuant LAS 4000 with ImageQuant LAS 4000 Biomolecular Imager software (GE Healthcare). The difference in the protein expression level between treated and untreated cells was analysed in at least 3 independent experiments.

Mouse neuroblastoma Neuro-2A (N2A) cells (ATCC CCL-131) were transfected with the indicated plasmids using PolyJet (SL100688, SignaGen Laboratories) according to the manufacturer’s instructions. For immunoprecipitation analysis, 1 day after transfection, transfected N2A cells were washed with PBS and solubilized in lysis buffer (PBS (pH 7.4), 1% Triton X-100, 2 mM PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstatin, and 10 μm MG132) at 4°C for 30 min. The lysates were centrifuged at 15,000×g for 30 min to remove the cell debris. The solubilized extract was subjected to immunoprecipitation using Myc or HA antibodies combined with protein A sepharose. After mixing by rotation at 4°C overnight, the precipitates were sequentially washed twice with each of the following buffers: 1% Triton X-100/PBS, 0.5% NP-40/0.5 M LiCl/50 mM Tris (pH 8), 0.5 M LiCl/50 mM Tris (pH 8), 10 mM Tris (pH 8). The precipitated proteins were then separated using SDS-PAGE and analyzed by immunoblotting. The immunoblotting results were recorded using an ImageQuant LAS 4000 system with ImageQuant LAS 4000 Biomolecular Imager software (GE Healthcare) and quantified using ImageJ/FIJI software. Relative expression levels of KLHL17 were normalized against HSP90 or actin. The uncropped full-size blots are available in S1 Raw Images.