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2 protocols using flag m2 beads

1

Protein Purification from Bacterial Cultures

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Protein expression was induced overnight in S. Typhimurium SL1344 strain at 22°C or in E. coli BL21 (DE3) at 16°C with 0.4 mM Isopropyl β -D-1-Thiogalactopyranoside (IPTG) after OD600 reached 0.8–1.0. The cells were collected by centrifugation at 4,000 g for 20 min and lysed by sonication. The lysates were centrifuged twice at 12,000 rpm at 4°C for 30 min to remove insoluble cell fragments. According to the corresponding methods, the supernatants of Flag tag proteins, 6x His tag proteins, and GST tag proteins were purified with Flag M2 beads, Ni-NTA agarose (GE Healthcare), and glutathione sepharose (GE Healthcare), respectively (Li et al., 2013 (link)). For GlcNAcylated-PhoP, His-PhoP was co-expressed with GST-SseK3 in E. coli BL21 (DE3) and purified with Ni-NTA agarose (GE Healthcare) as mentioned above. The protein concentration was examined by SDS-PAGE with BSA standards, followed by Coomassie Blue staining.
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2

Immunoblotting and Coimmunoprecipitation Assays

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For immunoblotting, cells were lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific), Dithiothreitol, and a protease/phosphatase inhibitor cocktail (Cell Signaling Technology). For coimmunoprecipitation assay, cells were lysed in 50 mM Tris, pH 6.8, 0.5% NP-40, 200 mM NaCl, 10% glycerol, 1 mM EDTA, and 1× protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Cell lysates were incubated with Flag-M2 beads for 2 h at room temperature or with V5 antibodies for 2 h at 4°C, then incubated with Protein A Sepharose 4 Fast Flow and Protein G Sepharose 4 Fast Flow beads (GE Healthcare) for 1 h at 4°C. Immunoprecipitates were subjected to Western blotting. The antibodies used were directed against STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 689 STAT2 (Millipore), ISG15 (Santa Cruz Biotechnology), mAb cl.2.1, a gift of E.J. Borden, Cleveland Clinic, Cleveland, OH), V5 (Invitrogen), and Flag (Sigma-Aldrich). Antibodies to phospho-Tyr 701 STAT1, USP18, β-Actin, IFIT1, and AKT were all from Cell Signaling Technology. The chemiluminescence detection reagent was Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or Western Lightning Plus-ECL (PerkinElmer).
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