V5 miniturbo nes pcdna3

Several biotin ligase constructs utilized for this study were obtained from Addgene, including MCS-BioID2-HA (plasmid no.: 74224), FLAG-TurboID (plasmid no.: 124646), and V5-miniTurbo-NES_pCDNA3 (plasmid no.: 107170). Plasmids were transfected into cells using XtremeGene HP DNA transfection reagent (Roche, Applied Science) according to the manufacturers' protocols.

Human ARIH1 ORF was cloned into V5-miniTurbo-NES_pCDNA3 (Plasmid #107170 Addgene), a gift from Alice Ting [45 (link)]. Hek293 cells were transiently transfected with either empty vector miniTurboID or ARIH1-miniTurboID and treated with 100 μM biotin (Sigma) 3 h prior to harvesting in RIPA lysis buffer. Lysates were incubated with streptavidin agarose beads (Invitrogen) overnight at 4 °C with rotation. Beads were washed three times in RIPA buffer and proteins were eluted by heat denaturation at 95 °C for 5 min in Laemmli buffer. Mass spectrometry analyses were performed by the Taplin Biological Mass Spectrometry Facility at Harvard Medical School.

The biotin ligase constructs, MCS-BioID2-HA (#74224) and V5-miniTurbo-NES_pCDNA3 (#107170), were obtained from Addgene and conjugated to the carboxyl-terminal of IFNLR1. HEK-293T were transfected with these constructs and then treated with 50 μM biotin (Fisher Scientific) for 1 h. Media was replaced with biotin-free media for the final 15 to 30 min. Cells were washed with cold 1× PBS on ice twice, and cells harvested with cold buffer (PBS with 1% Triton X-100, 0.2% SDS, and protease inhibitor (Thermo Scientific)). Samples were rotated at 4 °C for 20 min and then sonicated for 30 s at 25 °C three times. Streptavidin magnetic beads (Pierce) were magnetically sorted and rinsed with wash buffer 1 (PBS with 1% Triton X-100, 0.2% SDS) twice. Beads were then added to samples and rotated at 4 °C for 2 h. Beads were rinsed with wash buffer 1 twice, then rinsed with wash buffer 2 (8M urea in 50 mM Tris made in 1xPBS) twice more. Beads were rinsed again with wash buffer 1 then 1× PBS. Finally, 4× SDS + biotin mix (4× Laemmli dye (Bio-Rad) + 10% BME (VWR) and 50 μM biotin in wash buffer 1) was added to the beads. Proteins were eluted by heating at 95 °C for 5 min and run on a 4 to 20% precast gels (Bio-Rad). The lane was then excised and sent to the Biomedical Mass Spectrometry Center (University of Pittsburgh) for further purification and mass spectrometric analysis.