F79 500

Microtissues were fixed within the agarose-molded wells of the tissue culture plate with cold PFA solution (4%(w/v) paraformaldehyde, 8% (w/v) sucrose) (F79-500, Sigma-Aldrich) (S7903, Sigma-Aldrich). Microtissues were fixed for 2 h followed by 3 PBS washes. Following several PBS washes, the agarose mold was carefully removed from the bottom of the plate well and the excess gel was cut away while remaining cautious to not cut too close to the tissue itself. The remaining agarose mold containing the microtissue was then placed in warmed PB and heated on a hot plate at 200 °C for 10 min with frequent agitation until the agarose had melted, leaving behind the freely floating fixed microtissue. The microtissue was then transferred to a 35 mm petri dish and washed several times with warmed PBS to remove any residual agarose before transfer to a 48-well plate. Microtissues were left in cold PBS until immunohistochemical labeling was performed.

In 20 μl, 3 μg recombinant His6-MBP or MBP-eIF2A-His6 was mixed with 20% RRL in 100 mM KCl, 10 nM amino acid mix minus methionine, 10 nM amino acid mix minus leucine, and 0.5 mM MgOAc (same as in vitro translation above) or with 0.27 μM purified 40S ribosomal subunits in 40S Binding Buffer (14 (link)) (final of 50 mM HEPES, 100 mM KCl, 5 mM MgCl₂,; pH 7.5) and were incubated at 25°C for 10 min. Indicated samples were then crosslinked with 0.25% (v/v) formaldehyde (Sigma # F79-500) for 30 min at 25°C and quenched with 20 μl 100 mM Tris–HCl, pH 7.5. Samples were diluted 1:10 in ice-cold Wash Buffer (20 mM Tris–HCl, 140 mM KCl, 10 mM MgCl₂, 0.1% (v/v) Triton X-100, 10 mM imidazole; pH 7.5) and added to 20 μl HisPur Ni-NTA Magnetic Beads (Thermo Fisher # 888831) for 30 min at 4°C with end-over-end rotation. For 40S ribosomal subunit and 80S ribosome binding experiments, HisPur Ni-NTA Magnetic Beads were blocked with 1 μg/μl BSA (Invitrogen # AM2616) and 2 μg/ml yeast tRNA (Thermo Fisher # AM7119) in Wash Buffer for 1 h at 4°C with end-over-end rotation. Beads were then washed 5× with 400 μl ice-cold Wash Buffer. Bound proteins were then eluted with 200 μl 2× reducing LDS sample buffer (BioRad # 1610747) and heating for 15 min at 70°C. 20 μl of eluate was analyzed by SDS-PAGE and Western blotting as described above.