Immobilion fl membrane

All cell lysates and immunoprecipitations were analysed by LI-COR quantitative infrared western technology. Proteins were separated by SDS–PAGE and blotted onto Immobilion FL membrane (Millipore). Membranes were incubated with indicated primary antibody and subsequently with IRDye 800 or 680 secondary antibodies (LI-COR). Membranes were scanned using the Odyssey Sa imaging system (LI-COR) and quantification was carried out using the Odyssey Sa Application software (LI-COR). Uncropped scans of key western blots are found in Supplementary Fig. 3.

Following SDS–PAGE separation, gels were blotted onto Immobilion FL membrane (Millipore). Membranes were incubated with the indicated primary antibody and subsequently with IRDye 800 or 680 secondary antibodies (Li-Cor). Membranes were scanned using the Odyssey Sa imaging system (Li-Cor) and quantification was carried out using the Odyssey Sa Application software (Li-Cor). Representative images from at least two independent experiments is shown in all figures.

Dulbecco’s modified Eagle’s medium (DMEM) culture media, glutamine, gentamicin, and antibiotic–antimycotic solution were purchased from Life Technologies Corp. (CA, USA). Collagen type IV, HEPES, dl-dithiothreitol (DTT), pig insulin, sodium bicarbonate, glucose, sucrose, l-proline, d-(+)-galactose, NaCl, trizma base, trizma hydrochloride, and glycine were obtained from Sigma-Aldrich (MO, USA). Acrylamide, N,N′-methylene-bis-Acrylamide, N,N,N′,N′-tetramethylethylenediamine (TEMED), ammonium persulfate, Tween 20, protein standard of molecular weights, and the protein determination kit, based on the Bradford assay (Cat. 5000-002), were purchased from Bio-Rad (CA, USA). Amicon YM-3 filters and Immobilion-FL membrane were obtained from Millipore (MA, USA), fetal bovine serum (FBS) from Equitech (England), and ECL plus Western blotting detection systems from General Electric Healthcare (Buckinghamshire, UK).