FIG 9.

ORF57 association with polysomes enhances PABPC1 binding to, but prevents Ago2 association with, polysomes by polysome profiling of HEK293T cytoplasmic fractions. HEK293T cells were transfected with or without an ORF57-expressing vector (pVM7). At 24 h posttransfection, cytoplasmic fractions (after nucleus removal) were analyzed for polysome profiling across a 4.5% to 45% sucrose density gradient. The A₂₅₄ absorption spectrum of the fractions was automatically collected using a fractionator attached to the spectrophotometer to show a distinct pattern of peaks of free RNPs, monosomal 40S, 60S, and 80S, and polysomal complexes (A). The input and collected sample fractions (2 to 10) were used for RNA extraction and rRNA detection by 1.5% agarose gel electrophoresis using ethidium bromide staining (B). Total protein from each fraction was immunoblotted for detection of PABPC1, Ago2, ORF57, TIA-1, and PYM by the corresponding antibodies (C). Panels show one representative of three independent experiments. RNPs, ribonucleoproteins.