Cells infected with IAV 24 h post-transfection were harvested using standard lysis buffer supplemented with 1x protease inhibitor cocktail (Sigma Aldrich). 1/10 of the lysate was taken as input control. Immunoprecipitation was done using M2 flag affinity beads (Sigma) by incubating overnight with the lysate followed by multiple washes as per the manufacturer's instruction. The RNA was isolated from the input, and the immunoprecipitated fraction using the TRIzol reagent and cDNA was made. Further, the pull-down viral RNA expression was quantified using qPCR. Fold differences in viral RNA levels relative to RIG-I were determined using the following equation: (Thapa et al., 2016 (link)).
M2 flag affinity beads
Manufactured by Merck Group
M2 FLAG affinity beads are a purification tool used for the isolation and detection of FLAG-tagged proteins. These beads are coated with a monoclonal antibody specific to the FLAG epitope, allowing for the efficient capture and recovery of FLAG-tagged target proteins from complex samples.
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5 protocols using m2 flag affinity beads
1
Quantifying IAV RNA Binding Proteins
2
Immunoprecipitation of HSF Complexes
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hsf1−/−hsf2−/− MEFS were transfected with HSF1-HA alone, or with WT-HSF2-3xFLAG or HSF2ΔLZ1-3-3xFLAG for as described above. Cells were crosslinked with 1.5 mM DSP (Pierce) for 30 min at room temperature according to manufacturer’s protocols. Cells were lysed in SDS lysis buffer prior to sonication 1 × 30 sec to disrupt chromatin associated HSF complexes. 1.5 mg of total protein was then immunoprecipitated using M2 FLAG affinity beads (Sigma) overnight at 4°C. Beads were then washed 3× in lysis buffer prior to elution with TE buffer + 1% SDS. HSF1-HA protein was detected with anti-HA antibody (Y-11, Santa Cruz) (Supplementary Data Set 3 ).
3
Immunoprecipitation of HSF Complexes
4
Analyzing DDR1 Signaling upon Collagen Stimulation
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HEK-Flag-TurboID and HEK-DDR1-Flag-TurboID cells treated with vehicle or collagen I for 2 hours were lysed (Cell Signaling Technology lysis buffer), sonicated, clarified by centrifugation, and then equal amounts of cell lysates (200 μg) from HEK-turboID or HEK-DDR1-turboID were precleared by incubation with protein A for 1 hour at 4°C, and then incubated with Flag M2 affinity beads (Sigma-Aldrich) overnight at 4°C. The next day, Flag-beads were washed with 50 mM Tris pH 7.5 containing 150 mM NaCl and 1% Triton X-100, eluted in sample buffer (62.5 mM Tris pH 6.8, 2% SDS, 10% glycerol), and analyzed by Western blot for phosphorylated and total DDR1 and for total BCR.
5
Mapping NSun3-RNA Interactions in HEK293T
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A covalent bond between NSun3 protein and RNA in HEK293T cells expressing NSun3.FLAG.STREP2 was induced by irradiation with ultraviolet (UV) light (250 mJ cm−2). After cell collection, mitochondria were extracted by differential centrifugation. Pelleted mitochondria were lysed and RNA was partially digested with 4 μl micrococcal nuclease (NEB) for 15 min at room temperature in the presence of 3 μl TURBO DNase. The reaction was stopped by adding 12 μl SUPERasin (ThermoFisher Scientific). After immunoprecipitation with FLAG M2 affinity beads (Sigma) and elution with FLAG peptide as recommended by the manufacturer, proteins were digested with 4 mg ml−1 Proteinase K. RNA was end repaired with Antarctic Phosphatase and T4 Polynucleotide Kinase. Library preparation and high-throughput sequencing were similar as for BS RNA-Seq.
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