iProXProteomicsYuning SunHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0016588000sunyuning1994@nxmu.edu.cnNingxia Medical UniversityiProXfalseMass spectrometry analysis of proteins interacting with HBoV1 NS1, NS2, and NS1-70HEK293T cells in 10 cm dishes were transfected with 10 μg of HA-tagged viral protein plasmid, and total protein was collected 48 h after transfection. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 2 mM DTT, 100 μM PMSF, 1 μg/mL protease inhibitor cocktail) on ice for 30 min. Primary antibodies were mixed with supernatants of cell lysates (2 μg primary antibody per 1 mg protein sample) overnight at 4˚C and then incubated with Dynabeads™ Protein G/A for 4 h at 4˚C. Beads were washed 3 times with wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM EGTA), 3 times with high-salt wash buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.1% Triton X-100, 1 mM EGTA), and 3 times with 1× PBS to eliminate non-specific protein binding. After retaining 1/10 of the beads for western blot validation of IP efficiency, the remaining beads were sent to SpecAlly Life Technology Co., Ltd. for label-free quantitative mass spectrometry (MS) analysis.Sat Apr 11 00:00:00 BST 2026PXD0770569606