m7GTP binding assay

Jurkat cells were cultured in RPMI media, supplemented with non-essential amino acids (Gibco) and sodium pyruvate (Gibco). Jurkat cell pellets were lysed using Buffer B (10 mM HEPES pH 7.4, 1 mM MgCl₂, 10 mM NaCl, 50 mM NaF, 0.5% NP-40). Lysates were pre-cleared with Protein A-Sepharose beads (Generon, PCA-125) for 30 min at 4°C, rotating. Lysate were split into equal parts, each incubated with 40 ul ^m7GTP-Agarose (Jena Bioscience) or GTP-Sepharose for 2 h at 4°C. Beads were washed with Buffer B. After washes, beads were re-suspended in LDS sample buffer (Novex) with 0.1 M DTT diluted in Buffer B. ^m7GTP and GTP pulldown samples were resolved on the SDS-PAGE gels with SDS Running Buffer (25 mM Tris, 250 mM glycine, 0.1% SDS), then transferred to PVDF membranes, which were probed with rabbit anti-LARP1 antibody (ProteinTech).