Fig. 8. Stau2⁵⁹ and Stau2⁵² isoforms are associated with ribosomes. (A) Cytoplasmic extracts from rat cortical neurons in culture were prepared, placed on a 20-60% discontinuous sucrose gradient and centrifuged at 175,000 g for 3 hours. Fractions were recovered and analyzed by western blotting with monoclonal anti-Stau2 (Stau2), anti-L7a (L7a) and anti-calnexin (CNX) antibodies. The same results were obtained from three independent experiments. R, ribosomes. (B) Cytoplasmic extracts from neurons in culture were treated with 25 mM EDTA to dissociate ribosomal subunits and centrifuged at 250,000 g for 4 hours on a 10-40% continuous sucrose gradient. Fractions were recovered and analyzed by western blotting with monoclonal anti-Stau2 antibodies. Similar results were obtained with rabbit polyclonal anti-Stau2 antibodies (data not shown). The position of the 40S and 60S ribosomal subunits was determined with a spectrophotometer set at 254 nm.