Amersham typhoon 5 biomolecular imager

For each reaction, 100 nM of the indicated DNA substrate was incubated with the indicated concentration of MGME1 in a reaction buffer containing 100 μg/ml bovine serum albumin, 10 mM HEPES (pH 7.4), 150 mM NaCl and 2.5 mM MgCl₂ at 37°C. For the time-course experiments, an aliquot of the reaction mixture was pipetted out at the indicated time points and quenched by mixing with an equal volume of 2× TBE/urea sample buffer (Thermo Fisher Scientific) and heating at 65°C for 20 min. To analyze DNA cleavage patterns, quenched reactions were separated by 20% native TBE polyacrylamide gel electrophoresis. The results were imaged using an Amersham Typhoon 5 Biomolecular Imager (Cytiva), with a 473 nm laser and 525BP20 filter for 6-carboxyfluorescein (FAM), a 635 nm laser and 670BP30 filter for Cy5, and a 532 nm laser and 570BP20 filter for Cy3. The band signals were quantified using ImageJ. Statistical significance (P values) was determined in GraphPad Prism, version 9.5.1 for MacOS (GraphPad Software, Boston, MA, USA, www.graphpad.com) by two-tailed paired t-test, with ** indicating P < 0.01.

The kinetics with which a capped rpl41a mRNA was recruited to PICs was determined using a previously described native gel electrophoresis-based assay to monitor the mobility of a [³²P]-cap-labeled rpl41a mRNA^{22 (link),36 (link),53 (link),54 (link)}. A 2× PIC Master Mix was formed by combining 2 µM eIF1, 2 µM eIF1A, 600 nM eIF2, 750 nM Met-tRNA^iMet, 4 µM eIF4A, 800 nM eIF4B, 100 nM 40S subunit, and 4 mM ATP•Mg²⁺. This 2× PIC Master Mix was then used to generate a 1× reaction by adding stock solutions of eIF3_R or eIF3_N to the final specified concentrations. mRNA recruitment reactions were initiated by adding [³²P]-cap-labeled rpl41a mRNA to a final concentration of 15 nM. mRNA recruitment reactions were incubated continuously at 26 °C. At each specified time point, 4 µL of the reaction was removed and combined with 1 µL of Native Loading Dye and loaded onto an actively running 4 % polyacrylamide (37.5:1 acrylamide:bis-acrylamide) gel prepared in THEM Buffer. This gel was then run for 70 min at 200 Volts, and maintained at 16 °C with a circulating water cooler^{22 (link),37} . Subsequently, the gel was exposed to a phosphor screen overnight and then imaged on an Amersham Typhoon 5 Biomolecular Imager (Cytiva, Catalog Number 29187191).