Ha peptide

HEK293T TLR2ectodomain-HA, HEK-Blue™ Null1 TLR2-RRQQ, and HEK-Blue™ Null1 TLR2-QQQQ cells were lysed using RIPA lysis buffer (Merck Millipore, Billerica, MA, USA) with a protease inhibitor cocktail (Sigma, St. Louis, MO, USA) at 4°C for 10 min followed by two times sonication (Bandelin, Berlin, Germany) for 5 s at 50% power. Supernatant was collected after centrifugation at 14,000 g for 10 min. HA tagged proteins were immunoprecipitated using Pierce^® Anti-HA Agarose in a microcentrifuge tube. Protein was eluted using HA peptide (Thermo Scientific, Waltham, MA, USA) by incubating two times for 15 min with single bed volume of HA peptide at 30°C. Isolated protein was desalted and HA peptide was removed using Zeba Spin Desalting Columns and Devices, 40 K MWCO (Thermo Scientific, Waltham, MA, USA). Isolated desalted protein was quantified using the Thermo Scientific BCA protein assay kit (Thermo Scientific, Waltham, MA, USA).

For mass spectrometry, cytoplasmic extracts were obtained as aforementioned. Tandem immunoprecipitation (Flag and HA) was carried out using 10 mg of cytoplasmic extract. Flag IP was performed using EZview™ Red ANTI-Flag® M2 Affinity gel (SigmaAldrich, F2426), following the manufacturer’s instructions. Washes were carried out 3 times as aforementioned and protein complexes were eluted by competition performing 2 consecutive elutions using Flag elution buffer (250 ng/μl FLAG® Peptide (SigmaAldrich, F3290), diluted in IP buffer) incubating for 1 h at 4°C on a rotating wheel. HA IP was performed using the elutions obtained from the first IP incubated with Pierce™ Anti-HA Agarose beads (ThermoScientific, 26181) for 2 h at 4°C on a rotating wheel. Washes were carried out 5 times as aforementioned and elutions were performed using HA elution buffer (400 ng/ μl HA peptide (ThermoScientific, 26184), diluted in IP buffer) for 1 h at 4°C on a rotating wheel. Following elution, beads were removed using Pierce™ Centrifuge Columns (ThermoScientific, 11894131), as specified by manufacturer’s instructions. Silver-staining was performed according to the manufacturer’s instructions (Silverquest, Invitrogen). Mass spectrometry was performed at Taplin facility, Harvard University, Boston, MA.

All lysates were incubated with Pierce™ Control Agarose Matrix (ThermoFisher) for 20 min at 4 °C in order to remove any non-specific binding, following by an incubation with pre-washed AntiHA.11 Epitope Tag Affinity Matrix (BioLegend) for 4 hours (in vitro samples) or overnight (in vivo samples) at 4’C. Tagged ribosomes were then eluted from the beads by incubating with 200 µg/mL HA peptide (ThermoFisher Scientifc) for 15 min at 30’C with constant agitation. Non-protected RNA was digested with 10 ul of RNase I (ThermoFisher Scientific) for 40 min at 25 °C and the reaction was stopped by adding 13 ul of SUPERASE (ThermoFisher Scientific). Ribosome protected fragments (RPFs) were finally purified using miRNeasy minikit (Qiagen) accoridng to manufacturer’s instructions.