Salt extraction of polyribosomes releases siRNAs as a 70-kD ribonucleoprotein. (A) Outline of the cell fractionation procedure. Numbers in parentheses refer to lane numbers in panel B. (B) Northern blot with the MT40 sense riboprobe of small RNAs from equivalent amounts of postnuclear supernatant (lane 1), S100 fraction (lane 2), ribosomal pellet (lane 3), soluble fraction from the ribosomal pellet extracted with 400 mM potassium acetate after spinning at 100,000g for 1 h (lane 4), and from the salt-washed ribosomal pellet (lane 5). (C) The soluble salt-extracted material from the ribosome pellet shown in lane 4 of panel B was loaded on a Superdex-200 column, and selected fractions were Northern-blotted with a MT40 sense riboprobe. (D) The membrane shown in C was stripped and rehybridized with a sense INGI riboprobe. (E) An aliquot of the S100 fraction was loaded on a Superdex-200 column, and RNA from selected fractions was Northern-blotted with a MT40 sense riboprobe. The elution positions of carbonic anhydrase (30 kD), bovine serum albumin (66 kD), and thyroglobulin (669 kD) are indicated.
