For Western immunoblotting, RIPA lysates were harvested from porcine or human TM cells treated with 1 mM 4MU or vehicle control for 3 days, or from TM cells infected with the individual HAS silencing lentivirus. For infection, approximately 106 pfus silencing lentivirus were added to the media at the time of plating with 6 μg/mL polybrene. TM cells were infected for 6 days, cells and ECM were scraped into RIPA buffer and 30 μL of RIPA lysate was loaded into each lane. Proteins were separated on 10% SDS-PAGE gels (BioRad Labs, Hercules, CA) under reducing conditions and transferred to nitrocellulose. Primary antibodies were a monoclonal anti-HAS1 (Novus Biologicals, Littleton, CO), a goat anti-HAS2 polyclonal (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a rabbit anti-HAS3 polyclonal (Abcam, Cambridge, MA). Secondary antibodies were IRDye 700-conjugated anti-rabbit and IRDye 800-conjugated anti-mouse or anti-goat (Rockland Immunochemicals, Gilbertsville, PA). Western immunoblots were imaged using the Odyssey infrared imaging system (Licor, Lincoln, NE). Bands were quantitated using Image J software following background correction and values were then normalized to actin as a loading control. There was no significant difference in actin levels or total protein levels, as quantitated by a BCA assay (Pierce, Rockford, IL), between treatments and controls. Average pixel intensity was determined from 3 independent experiments.