Anti flag m2 a2220

Protein concentrations were measured using Pierce™ 660 nm Protein Assay Reagent (Thermo Scientific) or the Bradford protein assay (Bio-Rad). For immunoprecipitation, 2–3 mg of sNASP-FLAG-myc and sNASP-Strep-HA extracts were incubated for 3 hours at 4°C degrees with 50 μl of anti-Flag M2 (A2220, Sigma) or Anti-TwinStrep Mag-Strep beads (2–4090-002, Iba). After incubation, the beads were washed five times in ice-cold wash buffer (150 mM NaCl, 0.02% Nonidet P40, 50 mM Tris-HCl pH 7.6, 0.1 mM EDTA, 5% glycerol). Bound proteins were eluted with either Laemmli sample buffer 1X (LSB 4X: Tris-HCl (200 mM, pH 6.8), SDS 4%, glycerol (40%), DTT (100 mM)) or biotin elution buffer 1X (BXT buffer, 2–1042-025, Iba), respectively. Western blotting was performed as described previously (11 (link)). Western blots from three independent biological replicates were quantified using the software ImageJ, version 1.0.

HEK293T cells were lysed in lysis buffer containing 0.2% NP-40, 20 mM Tris-HCl pH 7.4, 150 mM NaCl, supplemented with protease inhibitors (cocktail from Roche) and phosphatase inhibitors (Naf, Na₄P₂O₇ and Na₃VO₄). Lysates were precleared for 30 min with Sepharose beads (6B100, Sigma-Aldrich), then anti-FLAG agarose beads (Anti-FLAG M2 A2220 Sigma-Aldrich) were added, followed by incubation for 2 h at 4°C. The immunocomplexes were then washed three times with lysis buffer, resuspended in 4X Laemmli buffer and analyzed by Western Blot.