Cultured cells were washed with ice-cold PBS and re-suspended in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% octylphenyl-polyethylene glycol [IGEPAL], 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS] containing 1 X Complete, EDTA-free, protease inhibitor cocktail [Roche, Laval, QC, Canada]). Whole cell extracts were vortexed and then clarified by centrifugation at 12000 x g for 15 minutes. Soluble extracts mixed with 2 X sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, and 5% beta-mercaptoethanol) were electrophoresed on 8-15% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences) for one hour, and proteins of interest were detected by probing with the appropriate primary and secondary antibodies diluted in Odyssey blocking buffer containing 0.1% Tween 20. Protein bands were detected and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences). All immunoblots were scanned at a wavelength of 700 nm for detecting IRDye 680 labeled antibodies and at a wavelength of 800 nm for IRDye 800CW conjugated antibodies [95, 96] . Signal intensities were quantified by means of the Odyssey software version 3.0. Beta-tubulin was always used as a loading control and for normalizing protein expression. Media samples analyzed for secreted Spn4A variants were taken directly from cultured cells, mixed with 2 X sample loading buffer, and subjected to the described Western blot analysis. [47] ; furin buffer contains 100 mM HEPES, pH 7.5, 1 mM CaCl 2 , 0.5% Triton X-100 [54, 58] , 1 X Complete EDTA-free protease inhibitor cocktail), and 11.6 ng/ml SKI-1/ S1P or 2.4 ng/ml furin. The enzyme mixture was incubated at 30uC for 30 minutes, and the reaction was stopped with 12.5 mM EDTA (final concentration). After completion, products were resolved on a 10% SDS-gel. The high-molecular weight band (EI) was visualized as described above for Western blotting.