Anti ha affinity matrix beads

293T cells expressing FH-CRBN were established by lentiviral transduction. Cells (1 × 10⁸) were collected in 1% Triton lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1% Triton X-100) supplemented with protease/phosphatase inhibitor cocktail. The cell lysate was mixed with M2 FLAG agarose (Sigma, A2220) and incubated for 3 h. After washing the beads six times with 1% Triton lysis buffer, bound proteins (CRL4^FH-CRBN) were eluted with 0.1 mg/mL FLAG peptide. Lt-NES cells (5 × 10⁸) were extracted using 1% Triton lysis buffer containing protease/phosphatase inhibitor cocktail, and the extracts were combined with the CRL4^FH-CRBN complex and 0.1% DMSO or 10 μM pomalidomide. After 12 h of incubation, the mixture was incubated with Anti-HA-Affinity Matrix beads (Roche, 11815016001) for 3 h. After washing the beads five times with 1% Triton lysis buffer, bound proteins were eluted with sodium dodecyl sulfate (SDS) and subjected to SDS-PAGE following Coomassie Brilliant Blue (CBB) staining. Gels were sliced and sent to Medical ProteoScope Co., Ltd. Tryptic peptides were extracted from the slices and subjected to the Ultimate 3000 RSLCnano liquid chromatography (ThermoFisher Scientific) and the Q Exactive Orbitrap mass spectrometer (ThermoFisher Scientific). Data were analyzed by Proteome Discoverer (ver. 2.2) (ThermoFisher Scientific) and Mascot (Matrix Science).

HEK293T cells were maintained at 37C, 5% CO2 in DMEM (Invitrogen, catalog 11995073) with added 10% fetal bovine serum (Sigma, catalog F4135). Vif-HA and A3-V5 plasmids were complexed with PEI-Max at a 2.5:1 ratio in Opti-Mem (Invitrogen, catalog 31985070) buffer for 30 min. Cells were transfected with plasmid: PEI complexes and harvested 48 h later, washed with PBS and lysed in lysis buffer (50 mM Tris, 75 mM NaCl, 0.1% NP-40 and Complete Protease Inhibitor Cocktail Tablet (Roche, catalog 04693159001) (pH 7.4)) at 4°C for 15 min, followed by centrifugation at 10,000 × g for 20 min. HA immunoprecipitation was carried out by mixing soluble lysates of transfected cells from a 10 cm dish with 40 uL anti-HA affinity matrix beads (Roche, catalog 11815016001) and incubating the mixture at 4°C for 3 h. The samples were washed six times with wash buffer (20 mM Tris, 50 mM NaCl, 0.1 mM EDTA and 0.05% Tween-20 (pH 7.5)). Bound protein was eluted with elution buffer (100 mM glycine-HCl, pH 2.5). The eluted protein was analyzed by SDS-PAGE and immunoblotting with appropriate antibodies. Infectivity (MAGI) assay was performed as previously described [11 (link)].