Anti flag m2 affinity agarose gel

For protein binding studies, 293T cells were seeded on 100 mm-diameter petri dishes and transfected with pcmCIITA (2 µg) alone or in combination with 2 µg of flag-TRIM22 or flag-ΔRING by using FugeneHD (Promega). Empty pcDNA3 vector was used as a stuffer DNA. Twenty-four hours after transfection, the cells were lysed on ice for 45 min with lysis buffer (1% NP-40, 10 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM EDTA) supplemented with 0.1% protease inhibitor cocktail (Sigma-Aldrich). Pre-cleared cell lysates were immunoprecipitated (IP) overnight at 4°C with the anti-FLAG M2 affinity agarose gel (Sigma-Aldrich). Precipitated proteins were resolved on 8% SDS-PAGE (polyacrylamide gel electrophoresis) and analyzed by immunoblotting with anti-CIITA and anti-FLAG M2 antibodies. Ten percent of total cell extract was used to detect protein expression by Western blotting (input).

Purified proteins (250 nM each) were mixed together on ice in IP buffer (30 mM Tris-Cl [pH 7.5], 150 mM NaCl, 3.5 mM MgCl₂, 0.1% igepal CA-630, 0.25 mM TCEP, 1 mM ATP, 5% glycerol; input sample) and incubated at 30°C for 15 min. Reactions were then incubated on ice for 5 min and supplemented with either anti-FLAG M2 affinity agarose gel (Sigma-Aldrich) for IP of Rad54 or Dynabeads Protein A (ThermoFisher Scientific) preincubated with anti-Sfr1, anti-Rad51, or anti-Rad52 antibodies (Haruta et al., 2006 (link); Kurokawa et al., 2008 (link)). Following mixing at 4°C for 1.5 hr, the beads were washed with IP buffer (400 µL x1) and eluted in 1x SDS loading buffer (IP sample). Proteins were then separated by SDS-PAGE and detected by immunoblotting with anti-Rad51 (rat 1:10,000; Argunhan et al., 2020 (link)), anti-Sfr1 (mouse 1:200; Argunhan et al., 2020 (link)), anti-FLAG (mouse 1:10,000; Sigma-Aldrich F3165), or anti-Rad52 (rabbit 1:10,000; Kurokawa et al., 2008 (link)) antibodies. FIJI software (Schindelin et al., 2012 (link)) was used for quantification of in vitro IPs. Briefly, background subtraction was performed by the rolling ball method and the signal for IP’d and co-IP’d protein was quantified. The co-IP’d signal was then divided by the IP’d signal and expressed relative to wild type. Graphs were prepared using GraphPad Prism (version 8).

Co-IP techniques coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to screen cellular proteins for their ability to interact with IBV 5a protein. Vero cells were seeded in 10-cm cell dishes, and when the cell density reached 80% to 90%, the cells in each dish were transfected with 20 μg of pRK5-5a-Flag or the empty vector pRK5-Flag, which was used as a control. At 36 h posttransfection, cells were collected and lysed. Host cellular proteins that interact with the IBV 5a protein were immunoprecipitated using an anti-Flag M2 affinity agarose gel (Sigma-Aldrich Corp., St. Louis, MO, USA), then identified by LC-MS/MS, and finally analyzed by GO annotation (43 (link)). The Gene Multiple Association Network Integration Algorithm (GeneMANIA, http://www.genemania.org/) was used to reveal physical and genetic protein-protein interactions for protein-protein interaction (PPI) network construction and visualization (44 (link)).

For each experimental condition, ST2 or HeLa cells were grown on 15‐cm dish. Cells were washed and incubated 30–60 min in serum‐free DMEM. In some experiments, cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 μM). Cells were stimulated in 10 ml serum‐free DMEM with SF‐IL‐17 (500 ng/ml) for indicated time points, solubilized in 1.5 ml 1% DDM containing lysis buffer and cleared by centrifugation (21,130 g, 30 min, 2°C). In control samples, 0.5 μg of SF‐IL‐17 was added post‐lysis. Cleared lysates were incubated with 10 μl of anti‐FLAG M2 affinity agarose gel (Sigma) overnight, washed 3× with 0.1% DDM containing lysis buffer and eluted by boiling in SDS sample buffer with 50 mM DTT.