Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific. RNAs were column-purified in accordance to the manufacturer’s instructions (RNeasy Mini Kit, QIAGEN), and efficient poly(A) tailing was evaluated by agarose gel electrophoresis. CREB3L2 and ATF4 translation reactions were incubated separately for 90 min at 30°C following vendor’s guidelines (Rabbit Reticulocyte Lysate System, Promega). CREB3L2 and ATF4 protein products were confirmed by immunoblotting in pilot experiments. Upon completion of the translation protocol, lysates (45 μl) containing CREB3L2 and ATF4 were mixed and incubated at 37°C for 30 min with gentle agitation (300 rpm for 5 s every minute) before immunoprecipitation, as previously described (71 (link)). Immunoprecipitation was carried out overnight at 4°C with gentle rotation in PBS supplemented with 0.1% NP-40 and protease inhibitors (cOmplete, EDTA-free Protease Inhibitor Cocktail, Roche), using magnetic beads conjugated with anti-HA or anti-V5 antibodies (anti-HA beads: PI88836, Thermo Fisher Scientific; anti-V5 beads: NC0777490, MBL International). Washing cycles were repeated five times with immunoprecipitation buffer. Complex elution was performed in 2× Laemmli buffer [130 mM tris-Cl (pH 6.8), 0.1 mM dithiothreitol, 20% (v/v) glycerol, and 4% SDS diluted in water] by boiling at 95°C for 5 min. Immunoblot detection of tagged CREB3L2 and ATF4: anti-HA (1:4000; ab9110, Abcam), anti-V5 (R960-25, Thermo Fisher Scientific), and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000; 60004-1-Ig, Proteintech), in conjugation with TrueBlot anti-rabbit IgG horseradish peroxidase (HRP) (1:1000; Rockland) or Superclonal anti-mouse IgG HRP (1:10,000; A28177, Thermo Fisher Scientific).
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