Endo H assay

Whole cell lysate (WCL) for BJAB ERAAP-dsRed clones B2 and D4 was made using RIPA lysis buffer (150 mM NaCl, 1% Triton X-100, 5% Na Deoxycholate, 1% SDS, 50 mM Tris pH 8 in water) with Protease inhibitors (Roche, 11836170001). WCL lysate was incubated on ice for 30 min and centrifuged at max speed for 10 min to remove debris. Cleared lysate was transferred to a new tube and anti-ERAAP L1 antibody (1:200 dilution) was added. Samples were incubated rotating overnight at 4 °C. The next day, ERAAP was immunoprecipitated by adding 40 ul of Protein A Dynabeads (Invitrogen, 10001D) to each sample and incubating an additional 2 hr. Using a magnet, beads containing ERAPP were captured. Beads were washed four times with 50 mM Tris, 150 mM NaCl in water. Endo H assay was performed following manufacturer’s instructions (New England Biolabs, P0702S). Following addition of Glycoprotein Denaturing Buffer, each sample was aliquoted into two tubes. 1 ul of EndoH (Endo H +) or 1 ul of water (Endo H -) was then added to each tube. Samples were incubated for 1 h at 37 °C. ERAAP’s glycosylation state was assessed by western blot. Membrane was probed using anti-ERAAP L1 antibody (1:000 dilution). Anti-ERAAP L1 was produced in the Shastri lab.