CERKL localizes to polysomes and compact mRNPs.

A) HeLa cells stably expressing CERKL WT-HA or an N-terminal fragment of the protein (CERKL 1-256-HA), generated as described in the Materials and Methods section, were lysed and polysomes were isolated by centrifugation in a 0.5-1.3-1.7-2.1 M discontinuous sucrose gradient. Seven 1 ml fractions (1–7) and the pellet (P) were analyzed by immunoblot using antibodies that recognize HA, the ribosomal protein RPS6. Actin was used as control. B and C) Similar experiments were carried out incubating the cell lysates with EDTA (15 mM) (B) or RNase A (100 µg/mL) (C) before centrifugation. Antibodies that recognize HA and the ribosomal proteins RPS6 and RPL26 were used. D–F) The pellet (D) and fractions 2 and 3 of the gradient (E and F) were treated with 25 mM (D and E) or 450 mM (F) NaCl, with or without RNase A (100 µg/mL) and subsequently centrifuged in a continuous sucrose density gradient (0.3–1.5 M) as described in Materials and Methods. The new fractions (1–11, in D and 1–10, in E and F) and the pellet (P) were immunoblotted with HA and RPS6 (D) or RNase A (E and F) antibodies. Below the Western blots, the concentration of RNA quantified at 260 nm is shown. G) The same experiment as in E and F, with fractions 2 and 3 treated with 30 mM EDTA or 1 mM puromycin at 25 mM and 450 mM NaCl before the continuous sucrose gradient. Ten fractions and the pellet were analyzed by immunoblot using antibodies that recognize HA.