Lumiglo reserve chemiluminescent substrate kit

Equal amounts of protein extracts were electrophoresed on 12% sodium dodecyl sulphate (SDS) polyacrylamide gel prior to transfer on Hybond ECL nitrocellulose membranes (GE Healthcare). Membranes were blocked overnight in Tris‐buffered saline containing 5% dry milk (TBS + 5% milk) and incubated with rabbit polyclonal antibodies directed against Arabidopsis eIF4E1 (diluted 1:2,000 in TBS + 5% milk) (Bastet et al., 2018), eIFiso4E (diluted 1:2,500 in TBS + 5% milk) (Estevan et al., 2014), and a mouse monoclonal antibody directed against plant actin (diluted 1:5,000 in TBS + 5% milk) (Sigma‐Aldrich). Following three washes with TBS supplemented with 0.1% Tween 20, membranes were probed with secondary goat horseradish peroxidase‐linked anti‐rabbit serum for detection of eIF4E1 and eIFiso4E (diluted 1:2,000 in TBS + 5% milk) and a goat horseradish peroxidase‐linked anti‐mouse serum for detection of actin (diluted 1:5,000 in TBS + 5% milk). Detection of horseradish peroxidase activity was performed using a LumiGLO Reserve chemiluminescent substrate kit (SeraCare) and X‐OMAT LS films (Kodak).

Cells were harvested and lysed in RIPA buffer supplemented with protease inhibitors (P-8340, Sigma-Aldrich, US). Protein concentrations in cell lysates were measured using the Pierce™ BCA protein assay kit (23225, Thermo Fischer Scientific, US), and the same amount of protein was loaded and separated on a polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane which was subsequently blocked for 2 h with a 2% BSA solution (prepared in TBST) and incubated overnight with primary antibodies against mouse c-Maf (sc-7866), Bach-1(sc-100995), Elmo-1 (sc-166661) and the loading control Gapdh (sc-365062). Membranes were then incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies directed against mouse (sc-2005) or goat (sc-2020) species (all antibodies purchased from Santa Cruz® Technology, CA, US). Membranes were incubated with LumiGlo Reserve™ chemiluminescent substrate kit (54-61-01, Sera care, Life Sciences, MA, US) and protein bands were visualized using the UVP Biospectrum™ 500 imaging system with the VisionWorks® images acquisition and analysis software (Analytik Jena, CA, US).