Identification of CTNNBL1 associating with viral IN

Published: 16 February 2022| Version 1 | DOI: 10.17632/rpb8zk3bmh.1
Guoxin Liang


Jurkat cells were infected with NL4-3-Luc (VSV-G) and lysed for IP assays with anti-IN or IgG antibodies (n = 3). IP products were further subjected by LC-MS assays to identify IN-interacting protein


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Jurkat cells (6.5 × 106) were infected with HIV-1NL4-3-Luc (MOI = 1.7). Twelve hours after infection, cells were washed with cold phosphate-buffered saline (PBS) twice and lysed with IP lysis buffer [50 mM Tris-HCl (pH 7.2), 50 mM NaCl, 1% NP-40, 1 mM EDTA, 2% glycerol, and 1× Complete]. Lysates were incubated on ice for 30 min and centrifuged at 12,000 rpm for 10 min at 4°C. Supernatants were transferred to fresh tubes, and the pellets were mixed with cold IP lysis buffer before sonication, followed by a second round of centrifugation at 12,000 rpm for 10 min at 4°C. Supernatants from the two extraction steps were pooled and incubated with mixed proteins A and G Dynabeads (Invitrogen) that had been pretreated overnight with antibody at 4°C. IP products were washed with cold IP buffer and PBS-Tween 20 (PBST) 5 to 10 times (500 μL/wash). Detergent removal and protein digestion used filter-aided sample preparation methods. IP products were first reduced in 20 mM dithiothreitol (DTT; Sigma) at 95°C for 5 min and subsequently alkylated in 50 mM iodoacetamide (IAA; Sigma) for 30 min in the dark at room temperature. After alkylation, samples were transferred to a 10 kDa centrifugal spin filter (Millipore) and sequentially washed with 200 ced in 20 mM DTT and 200 μL of 50 mM ammonium bicarbonate twice by centrifugation at 14,000 rpm. Trypsin (Promega) was added at 1:50 dilution (enzyme/substrate, M/M) in 200 μL of 50 mM ammonium bicarbonate at 37°C for 16 h. Peptides were recovered by transferring the filter to a new collection tube and spinning at 14,000 rpm. The filter was washed twice with 100 μL of 50 mM NaHCO3 to increase the yield of peptides. Peptides were desalted by STAGE TIP. MS experiments used a nanoscale ultra-high-performance LC (UHPLC) system (EASY-nLC 1000; Proxeon Biosystems, Odense, Denmark) connected to an Orbitrap Q-Exactive equipped with a nanoelectrospray source (Thermo Fisher Scientific, Bremen, Germany). Peptides were dissolved in 0.1% formic acid (FA) with 5% CH3CN and separated on a reverse-phase HPLC analytical column (75 μm  15 cm) packed with 2 m C18 beads (Thermo Fisher Scientific). Samples were eluted using a 2 h gradient from 5% to 40% acetonitrile (ACN) in 0.5% FA at 250 nL/min flow rate. A full MS/MS cycle consisted of one full MS scan in profile mode between m/z 300 and 1800. Subsequently, the 10 most intense ions were fragmented with high-energy collisional dissociation with normalized collision energy at 28% in centroid mode. The dynamic exclusion window was set at 40 s. Raw data were processed using Proteome Discoverer (PD; version 2.1), and MS/MS spectra were searched against the reviewed Swiss-Prot human proteome database. Two trypsin missed cleavages were allowed. Only peptides with at least six amino acids long were considered. Peptide and protein identification was filtered with PD to control the false discovery rate of <1%. At least one unique peptide was required for protein identification.


China Medical University