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. 2016 Aug 9;44(17):8335–8351. doi: 10.1093/nar/gkw702

Figure 4.

Figure 4.

Purified Gemin5 binds directly to the ribosome. (A) Diagram showing His-tagged Gemin5 polypeptides used in the ribosome-binding assays. Numbers indicate the amino acid residues flanking each polypeptide. (B) Binding assay of 80S ribosomes with His-Gemin5. 80S ribosomes (0.7 pmol) were incubated with the full length His-G5 (4 pmol) previously bound to Ni-agarose beads. After extensive washings, His-G5 was immunodetected using anti-G5 antibody. WB anti-P0 was used to determine the presence of ribosomes bound to Gemin5. (C) Binding assay of 40S and 60S ribosome subunits (0.7 pmol) with His-tagged Gemin5 (4 pmol). WB anti-P0 was used to determine the presence of 60S subunits bound to Gemin5, while anti-RACK1 was used to monitor the presence of 40S subunits. (D) Binding assay of 80S ribosomes with His-G51–739 or His-G51287–1508. Both, His-G51–739 or His-G51287–1508 were immunodetected using anti-His. WB anti-P0 was used to determine the presence of ribosomes bound to Gemin5. (E) Binding assay of 80S ribosomes carried out with the indicated mutants of His-G51–739. All binding assays were conducted independently at least three times.