Anti ha antibodies clone 3f10

Immunoprecipitation was performed as in Cohen et al. (2011) (link) with minor modifications. After growth in the different conditions described in the results, ∼10⁹ cells were harvested, washed in Milli-Q water and resuspended in lysis buffer [50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.6% Triton X-100, 10% glycerol, protease inhibitors (cOmplete Mini EDTA-free Protease Inhibitor Cocktail, Roche), and phosphatase inhibitors (Phosphatase Inhibitor Cocktail Set II, Millipore)]. Cells were lysed with glass beads in a cooled FastPrep (MP Biomedicals). Cell lysates were cleared by centrifugation at 13,000 × g for 30 min and supernatants were incubated for 30 min with 2.5 µg/sample anti-HA antibodies (clone 3F10; Roche) bound to 50 µl/sample (or 1.5 mg/sample) protein G-coated magnetic beads (Dynabeads Protein G; Invitrogen). The subsequent steps of immunoprecipitation and washes were performed according to the manufacturer’s instructions. The immunoprecipates were eluted in 2× Laemmli buffer.

Transient expression and co-immunoprecipitation were carried out as described previously (Alazem et al. 2017 (link)). N. benthamiana leaves were collected 2 d after coagroinfiltration with pBin-3HA-mCherry expressing either CPL, CPS, or P19 (in the C-terminal mCherry protein tag) along with pBin-eGFP. Total protein was extracted from 2 g of leaves (6 leaves from 3 plants) in extraction buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 M MgCl₂, 5 mM DTT, and 5% v/v Nonidet P-40) with protease inhibitor cocktail (Roche). Immunoprecipitation was carried out as described previously (Alazem et al. 2017 (link)). For input samples, HA was detected using anti-HA antibodies (clone 3F10; Roche). For co-immunoprecipitated samples, mCherry was detected using an anti-mCherry antibody. Immunoprecipitated RNA was extracted from HA-beads by TRIzol, as described previously (Brosseau and Moffett 2015 (link)). An amount of 250 ng of precipitated RNA was used to detect siGFP by stem–loop PCR, as described above.