Polyvinylidene fluoride or nitrocellulose membrane

Serum-free OptiMEM media was harvested from co-culture wells and protein was precipitated using trichloroacetic acid (TCA). Briefly, CM was mixed with 10% volume of 2% deoxycholate, followed by 10% volume of 100% TCA. CM was spun at 1.6K RPM with tabletop centrifuge for 5 min and removed of DOC/TCA. The protein pellet was resuspended in 50 uL of 1.5M Tris-HCl (pH 8.8) and an equal volume of 2X Laemmli SDS sample buffer was added, which was followed by incubation at ~95°C for 10 min. Protein concentration was measured by RCDC assay. 100 ug of protein for each sample were run on denaturing 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene fluoride or nitrocellulose membrane (GE Healthcare). Ponceau S Red (Thermo Fisher Scientific) was used to stain the proteins on the membrane to confirm equal loading of total proteins in gel lanes. Membranes were incubated with appropriate antibodies, analyzed via enhanced chemiluminescence (GE Healthcare), and quantified via ImageJ analysis (National Institutes of Health) to calculate the signal intensity of protein(s) of interest normalized to total Ponceau S Red stain protein quantified per lane.