Figure 1.

NEMF Is Required for Listerin Recruitment to Stalled Ribosomes

(A) HEK293T cells treated for 30 min with DMSO (control), 50 μg/ml CHX, or 1 mM puromycin (puro) were lysed and the cytosolic extracts separated on 10%–50% sucrose gradients. Eleven fractions from top (fraction 1) to bottom (fraction 11) were analyzed for the distribution of Listerin. The immunoblots and their respective quantification are shown.

(B) The A₂₆₀ profiles of the sucrose gradients from (A) are shown. As shown in Figure S1A, fractions 5 corresponds to 60S, fraction 6 to 80S, and fractions 7–11 to polysomes.

(C) Immunoblotting of the samples from (A) for NEMF recruitment to ribosomes.

(D) Cytosolic extract from CHX-treated cells as in (A) were size fractionated on a high-resolution 10%–30% sucrose gradient and immunoblotted for the indicated components. The positions of 60S and 80S are indicated. L9 (uL6) and S16 (uS9) are small and large subunit proteins, respectively. T indicates total lysate. Asterisk indicates background band. The plus symbol indicates multiple fractions were pooled (e.g., 5+ is fractions 5–8).

(E) HEK293T cells transfected for 30 hr with mock or NEMF siRNAs were analyzed for Listerin recruitment to ribosomes as in (A). The inset shows Listerin and NEMF levels in the total cytosol extract. Knockdown of NEMF was to ∼25% of control levels.

See also Figure S1.