(A) Crude RRL lysate was size-separated on a 5–25% sucrose gradient. The fractions were subjected to SDS-PAGE and visualized by immunoblotting for BAG6, ASNA1, SGTA, and GET4. (B) Uncropped version of the gels shown in Figure 5B. (C) In vitro translation of SEC61B in the presence of 35S-methionine and 15 µM DHQZ36.1 in crude rabbit reticulate lysate (RRL). Completed lysate reactions were subjected to size fractionation by centrifugation in a 5–25% sucrose gradient and their individual fractions chemically crosslinked with 0.250 mM bismaleimidohexane (BMH). Crosslinked samples were resolved by SDS-PAGE and visualized by autoradiography. Adducts to SEC61B are denoted with ×. (D) 35S-methionine-labeled SEC61B, which has C-terminal V5 epitope, was translated in crude RRL lysate in the presence of 15 µM of the indicated compounds. Translation reactions were subjected to chemical crosslinking (XL) with 0.250 mM BMH. Non-crosslinked samples were directly analyzed by SDS-PAGE. Crosslinked adducts to the TA protein substrate or ASNA1 were analyzed after denaturing immunoprecipitation (IP) with α-V5 or α-ASNA1 antibodies, respectively. For crosslinked products to GET4, samples were subjected to a non-denaturing IP with α-BAG6 antibody, which maintains the BAG6/GET4 interaction. Samples were visualized by autoradiography. Adducts to SEC61B are denoted with ×. (E) Representative data for SGTA~TA protein complex formation. In vitro translation of model TA substrate STX5TMD in the presence of 35S-methionine in PURE system supplemented with recombinant SGTA (final concentration of ~14 µM). Completed reactions were subjected to size fractionation by centrifugation in a 5–25% sucrose gradient. Fractions were analyzed by SDS-PAGE and visualized by Coomassie blue staining (top) and autoradiography (bottom). Fractions 3, 4, and five were pooled together and used as source of SGTA~STX5TMD in Figure 5E. * is a component of the PURE system (Mateja et al., 2015). (F) The complex of 35S-methionine labeled SEC61B and SGTA (SGTA~SEC61B) was generated using the PURE system. The transfer reactions were assembled with recombinant TRC pathway components in the presence of 15 µM of the indicated compounds and subjected to chemical crosslinking (XL) with 0.250 mM BMH. Samples were directly analyzed by SDS-PAGE, and autoradiography to visualize TA protein crosslinks (top), or Coomassie blue stain to detect the input proteins (bottom). Adducts to SEC61B are denoted with ×.