Fig 1: Association of SLFN14 (WT/mut)-myc with ribosomes and ribosomal subunits resulting in rRNA endonucleolytic degradation in HEK293T cells. (A) Binding of SLFN14 (WT/mut)-myc to 80S monosomes, 60S, and 40S ribosomal subunits obtained after SDG centrifugation of HEK293T cell lysates with overexpressed one of SLFN14 (WT/mut)-myc forms. Assignment of 80S monosomes, 40S, and 60S subunits in gradient fractions was based on immunoblotting with anti-RPS19 and anti-RPL3 antibodies as exemplified by the EV control. Ribosomal fractions were concentrated and analyzed by immunoblotting with anti-SLFN14 antibodies. (B) Expression levels of SLFN14 (WT/mut)-myc in HEK293T cells assayed by immunoblotting with anti-SLFN14 and anti-GAPDH (control) antibodies. (C) Binding of recombinant SLFN14 (WT)-65 kDa and SLFN14 (K218E)-65 kDa to assembled 80S ribosomes assayed by SDG centrifugation and Coomassie staining. (D) rRNA degradation in SLFN14(WT/mut)-overexpressed HEK293T cells assayed by denaturing agarose/formaldehyde gel electrophoresis (n = 3 independent experiments). The asterisks indicate the main bands of rRNA fragments. Positions of the 28S rRNA and 18S rRNA are shown. (E) Integrity analysis of rRNA. (Left panel) Representative electrophoretic profiles of total RNA obtained using the Agilent 2200 TapeStation system from SLFN14(WT/mut)-overexpressed HEK293T cells. Arrows indicate the appearance of rRNA degradation fragments. (Right panel) representative data output gel-like images. The average RIN scores from three independent experiments are shown.