35s met

Protein import assay was done using intact chloroplasts isolated from 10- to 13-day-old pea seedlings and [³⁵S]-labeled proteins as previously described (Inoue and Potter, 2004 (link); Inoue et al., 2006 (link)). The radiolabeled proteins were synthesized using T_NT^® coupled reticulocyte lysate system (Promega) and T7 (for Toc75-IV and Tic22), T3 (for OEP80tr), SP6 (for Toc75, and OEP80) RNA polymerases with [³⁵S]Met (Perkin Elmer, Waltham, MA). Post-import fractionation of chloroplasts was done as described (Inoue et al., 2006 (link)). For post-import protease treatment, the chloroplasts containing the imported proteins were treated with thermolysin or trypsin (both are from Sigma-Aldrich Corp, St. Louis, MO) at a 1:1 mass ratio with the amount of chlorophylls incubated in the import reaction in import buffer with (for thermolysin) or without (for trypsin) 1 mM CaCl₂, respectively, for 30 min in the dark on ice (for thermolysin) or at room temperature (for trypsin). The protease reactions were quenched by adding EDTA to the final concentration of 5 mM (for thermolysin) or trypsin inhibitor at a 10:1 mass ratio of the inhibitor to the protease (for trypsin) in import buffer. For the energy-dependency assay, the reaction was done using translation products pre-treated with 50 U/mL apyrase (Sigma-Aldrich) at room temperature for 15 min.

We imbibed three biological replicates of 20 isolated embryos and 5 embryoless endosperms in 4 ml of sterile distilled water with 50 μCi of [³⁵S]-Met (PerkinElmer) at 30°C during 24 h in the dark. Samples were placed on filter papers to remove excess water and grinded with mortar and pestle using liquid nitrogen. Proteins were then extracted according to previously published protocols (Rajjou et al., 2006 (link)). To avoid measuring the non-specific incorporation of radioisotopes into contaminants, we purified the total soluble proteins. In addition, dead seeds (autoclaved seeds) were used as a negative control in order to measure the background level related to non-specific incorporation of [³⁵S]-Met. Finally, 10 μl of protein extracts were added to 5 ml of scintillation liquid cocktail [Ecolite(+), MP Biomedicals, France]. Radioactivity was finally measured (3 biological and 3 technical replicates) using a liquid scintillation analyzer (Tri-Carb 2810TR, PerkinElmer, MA, USA) set between 5 and 100 keV with 10 min integration per sample.

tRNA misacylation was measured using a microarray method described previously [1 (link)]. Briefly, human embryonic kidney 293T (HEK293T) cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (HyClone) at 37°C with 5% CO₂. Cells were grown on 10 cm plates to ~80% confluency. For pulse-labeling experiments, 0.5 mCi [³⁵S]-Met (Perkin-Elmer, Boston, MA) was added onto each plate and incubated at 37°C for 8 min. Cells were washed twice with 300 mM NaOAc/HOAc pH4.8/10 mM EDTA and then scraped from the plate. Cells were transferred to tubes and spun down. Pellets were resuspended in ice-cold 300 mM NaOAc/HOAc pH4.8/10 mM EDTA. Charged tRNAs were isolated using acetate-saturated phenol/CHCl₃ (pH 4.8). RNAs were precipitated using equal volume of isopropanol and finally dissolved in 10 mM NaOAc/HOAc pH4.8/1mM EDTA. RNA concentration was measured using Nanodrop 2000 (Thermo Scientific) and 20 μg RNA was used for array hybridization.

Equivalent amounts of IAV PR8-infected MDCK cells were pulse-labeled for 2 min with [³⁵S]-Met (PerkinElmer; Waltham, MA) at 37°C, chased at the same temperature, detergent lysed, and subjected to IP as described [14] (link). When indicated, tunicamycin (5 µg/ml) or a mixture of DMN (1 mM) and SWN (10 µM) (Calbiochem; Billerica, MA) were added to cells 30 min before radiolabeling and maintained throughout the radioactive pulse and chase periods. Immunocollected proteins were analyzed by SDS-PAGE and visualized by exposing dried gels to Carestream Kodak BioMax MR (Sigma-Aldrich) films. Gels were stained with Coomassie brilliant blue R (MP Biomedicals, LLC; Santa Ana, CA) prior to drying to ensure that the Abs were equally recovered and loaded in all lanes. Since equal amounts of IgG were added to each sample, this served as a loading control. IB studies were carried out as reported [62] (link).