Irdye 680rd anti rabbit igg

For Supplementary Fig. 5a, lysates for ribosome profiling were subjected to SDS-PAGE and immunoblotting. For Supplementary Fig. 6b, cells cultured in a 24-well plate were transfected with the pI.18-3xFLAG, pI.18-SFSVNSs-3xFLAG, or pI.18-SFSVNSs-c1 + 2-Ala-mut-3xFLAG plasmid, as described in the “Nascent peptide labeling by OP-puro” section. After 48 h incubation, the cells were treated with Tg for 0, 2, 4, and 6 h at 37 °C and lysed in 100 µl of RIPA buffer (Nacalai Tesque, 16488-34) with Protease Inhibitor Cocktail (Nacalai Tesque, 25955-24) and Halt Phosphatase Inhibitor (Thermo Fisher Scientific, 78428). The cleared lysates were subjected to SDS-PAGE and immunoblotting. Anti-LC3B (Abcam, ab48394, 1 : 1000), anti-FLAG M2 (Sigma, F3165, 1 : 1000), anti-β-actin mAb 6D1 (Medical & Biological Laboratories [MBL], M177-3, 1 : 1000), and anti-β-actin pAb (MBL, PM053, 1 : 1000) were used as primary antibodies. IRDye 800CW anti-rabbit IgG (LI-COR, 926-32211, 1 : 10,000), IRDye 680RD anti-mouse IgG (LI-COR, 925-68070, 1 : 10,000), and IRDye 680RD anti-rabbit IgG (LI-COR, 926-68071, 1 : 10,000) were used as secondary antibodies. Images were acquired with an ODYSSEY CLx (LI-COR) imaging system.

For MAT2A protein quantification, cells were harvested and lysed using RIPA buffer (1 % NP40, 0.5 % DOC, 0.1 % SDS, 50 mM TRIS pH 8.0, 80 mM NaCl, 50 mM NaF, 20 mM Na₄P₂O₇, 1 mM EDTA, 1 mM EGTA, and 1 Complete tablets Mini Easypack (Roche) for 10 mL). An equal protein amount was loaded on a 10% SDS gel with Laemmli buffer and PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific), and blotted on an Immobilon-FL PVDF membrane (Merck-Millipore). After blocking, primary antibodies (GAPDH, clone 14C10 from Cell Signaling, Danvers, MA, USA and MAT2A, polyclonal from Novus Biologicals, Centennial, CO, USA) and the secondary antibody (anti-rabbit IgG, IRDye 680RD, polyclonal from Li-Cor Biosciences, Lincoln, NE, USA) were used. Analysis was carried out at the Li-Cor Odyssey CLx using the Image Studio Software (both Li-Cor Biosciences) for documentation. Relative quantification was performed using glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) as the reference protein.

Whole cell lysates were prepared using 1x RIPA buffer [SDS 0.1 % (w/v), Sodium deoxycholate 0.4% (w/v), NP-40 0.5% (v/v), 100 mM NaCl, 50 mM Tris-HCl, pH 7.4], supplemented with 1:100 protease inhibitor cocktail (Sigma P8340), phosphatase inhibitor cocktail III (Sigma P004), and 2 U/ml Benzonase (9025-65-4). Protein lysate concentration was determined using Pierce BCA protein assay kit (23225). Lysates were denatured for 5 min at 97 °C with Laemmli buffer containing 5% β2-mercaptoethanol, and the equivalent of 30 μg of total protein was loaded per lane. Samples were resolved by 10% SDS-PAGE and transferred to nitrocellulose membrane using iBlot2 transfer device (IB21001). The membrane was probed with anti-ZFP36 mouse monoclonal (Origene #OTI3D10) (2 μg/ml) and anti-ZFP36L1 polyclonal (CST #BRF1/2) (32 ng/ml) antibodies. Proteins were detected by incubating membrane with anti-Mouse IgG IRDye800CM (Licor #926-32210) and anti-Rabbit IgG IRDye680RD (Licor #925-68071), and scanning the membrane with Licor Odyssey CLx using standard methods. GAPDH was detected using rabbit monoclonal anti-GAPDH (CTS #5174) at 1:1,000 and anti-Rabbit IgG IRDye680RD. Image analysis was conducted using ImageStudio Lite version 5.2, and normalized protein signal was calculated using standard methods.