Edta free halt protease inhibitor

Preparation of small (40S) and large (60S) human ribosomal subunits was adapted from refs. 45 (link) and 73 . Specific deviations implemented for the purification of polysome fractions from human tissue culture are described here. Cell pellets were resuspended in lysis buffer [20 mM Tris HCl (pH 7.5), 2.5 mM MgCl₂, 10 mM KCl, and 1 mM freshly prepared DTT] with the RNase inhibitor RNase Out (Invitrogen), EDTA-free Halt Protease Inhibitor (Thermo Scientific), and cycloheximide (Sigma) at 100 μg/mL (∼350 µM). The solution was incubated on ice for 10 min before centrifugation in a Microfuge 22R Refrigerated Centrifuge (Beckman Coulter) at 14,000 rpm for 10 min at 4 °C to pellet cell debris. The supernatant was loaded onto precooled 10–50% sucrose density gradients and spun at 35,000 rpm for 3 h at 4 °C in an Optima l-100 XP ultracentrifuge (Beckman Coulter). The gradients were then fractionated using a BR-186-1 Fractionator and a UA-6 UV/Vis detector (Teledyne Isco). Fractions corresponding to polysomes were collected and subsequently pelleted and dissociated into subunits according to ref. 73 . Pelleted subunits were resuspended with storage buffer [30 mM Hepes (pH 7.5), 15 mM MgCl₂, 50 mM NH₄Cl, 2 mM spermidine, 5 mM putrescine, 1 mM DTT, and 6% sucrose] for stable, long-term storage in liquid nitrogen.

For preparation of whole‐cell lysates, cells were cultured for 2 days in complete media and then serum‐starved for 24 h prior to harvesting, unless indicated otherwise. Cells were lysed in RIPA buffer, supplemented with EDTA‐free HALT protease inhibitors (Thermo Scientific, Rockford, IL, USA), and 10 mm sodium orthovanadate (Sigma). Equal amounts of cell lysate, as measured by Lowry DC reagent kit (Bio‐Rad, Hercules, CA, USA), were subjected to SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose. Proteins of interest were detected via chemiluminescence after exposure to primary antibodies against NME1 (610247; BD Biosciences, San Jose, CA, USA), fibronectin (FN‐15, Sigma) and beta‐tubulin (H‐235, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by isotype‐specific HRP‐conjugated secondary antibodies.