Image studio lite v4

Protein extracts from confluent endothelial cell cultures were harvested by scraping in ice-cold RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris HCl, pH 7.4 plus one tablet per 10 mL of Roche cOmplete™ Mini Protease Inhibitor Cocktail). Protein quantity was determined using the Pierce^TM BCA Protein Assay Kit according to the manufacturer’s instructions. Ten µg of each sample were mixed with 5X SDS sample buffer, heated to 95 °C for 5 min and separated by SDS-PAGE (8% resolving gel; 80 V for 20 min and 120 V for 1.5 h). Proteins were transferred to a nitrocellulose membrane (semi-dry blotting at 50 mA for 45 min), which was then blocked for 1 h in Rockland Blocking Buffer MB-070 from Rockland Immunochemicals Inc. (LubioScience GmbH, Zürich, Switzerland). Antibody incubation was done for 1 h at RT or 4 °C overnight. Signals were visualised using an Odyssey Infrared reader (Li-Cor Biosciences, Bad Homburg, Germany) and quantified via Image Studio Lite v4.0 (Li-Cor Biosciences, Bad Homburg, Germany). Background subtraction was done automatically. Protein size was referenced to a prestained protein ladder (Thermo Fisher Scientific, 26619). For calculation of the relative PECAM-1 signal intensity, PECAM-1 signals were normalised to the GFP signal from the LifeAct-GFP protein expressed by the pMBMECs.

To generate whole-cell lysates, cells were harvested in 1 ml PBS on ice, combined with their supernatants and centrifuged at 300g/5 min. Pellets were washed with ice-cold PBS (300g/5 min). Cells were lysed in NET-N buffer (100 mM NaCl, 10 mM Tris-HCl pH 8, 1 mM EDTA, 10% glycerine, 0.5% NP-40; plus cOmplete protease inhibitor Tablets (Roche) and phosphatase inhibitor cocktail 2 (Sigma)) for 30 min on ice, sonicated (10 s/20% amplitude) and centrifuged (18,800g/15 min/4 °C). Protein concentrations of resulting lysates were measured by Bradford assay. Detection of proteins was performed by SDS-PAGE and western blots using enhanced chemoluminescence and X-ray films (GE Healthcare) or Odyssey Infrared Imaging System (Licor). To perform densitometric analyses, ImageJ and Image Studio Lite V4.0 (Licor) were used. Uncropped western blot images of data shown in Figs. 1–8 can be found in Supplementary Figures 7–12.

A 15 µg aliquot of protein from total O. tauri cell lysates was run on a Novex NuPAGE 4–12% Bis-Tris by SDS–PAGE with PeppermintStick Phosphoprotein Molecular Weight Standards and a Spectra Multicolor Broad Range Protein Ladder (Thermo Fisher Scientific). The gel was fixed overnight (50% methanol, 40% ddH₂O, 10% glacial acetic acid), washed in ddH₂O, and stained with Pro-Q Diamond Phosphoprotein Gel Stain (Invitrogen, now Thermo Fisher Scientific, Loughborough, UK) in the dark at 25 °C following the manufacturer’s instructions. The gel was imaged on a Typhoon TRIO variable mode imager (GE Healthcare, Amersham, UK) at 532 nm excitation/580 nm emission, 450 PMT, and 50 µm resolution. Images were processed using ImageQuant TL software (GE Healthcare, Amersham, UK). The gel was re-used for protein quantification using SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific) following the manufacturer’s instructions and imaged using a UV transilluminator (Ultra-Violet Products Ltd, Cambridge UK). Protein and phosphoprotein bands were quantified using Image Studio Lite v 4.0 (LI-COR Biosciences, Cambridge, UK).

Whole cell lysates of cells, SDS-PAGE and Western blotting were done as described [69 (link)]. Membranes were probed with primary antibodies (see Suppl. Table S3). Anti-rb or anti-m-IRDye680 or -IRDye800 (Licor, Germany) second step antibodies, resp. were used and detected with the Licor scanner using the Odyssey software (Licor). For densidometric analysis Image Studio Lite V4.0 (Licor) was used.