Ab86146

The mice were euthanized by CO₂ inhalation and portions of the indicated organs or tumors were employed to paraffin embedding and H&E staining. IHC staining was performed according to standard protocols. Briefly, the samples were deparaffinized, rehydrated through an ethanol series followed by antigen retrieval with sodium citrate or tris–EDTA buffer according to antibody manufacturer’s instruction. Sections were blocked with 10% FBS in PBS for 60 min at room temperature and were incubated with 3% H₂O₂ in methanol for 10 min at room temperature to block endogenous peroxidase and then incubated with anti-METTL16 (1:250, HPA020352, Millipore Sigma), anti-eIF3a antibody (1:200; ab86146, Abcam) or anti-eIF3b antibody (1:200; sc-137214, Santa Cruz Biotechnology). IHC staining was performed with horseradish peroxidase (HRP) conjugates using DAB (550,880, Biosciences) detection. All the slides were captured by a Widefield Zeiss Observer 7 microscope.

HepG2 cells were seeded on an 8-well chamber slide (154534, Thermo Fisher Scientific). After three washes with PBS, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the cells were incubated with 1 × Permeabilization Buffer (00-8333-56, Thermo Fisher Scientific) for 15 min, followed by blocked with Duolink block solution for 1 h at room temperature and incubated overnight at 4 °C with the following antibodies: (1) mouse anti-Flag (F3165, Sigma-Aldrich) and rabbit anti-eIF3a (ab86146, Abcam); (2) rabbit anti-METTL16 (HPA020352, Sigma-Aldrich) and mouse anti-eIF3b (sc-137214, Santa Cruz Biotechnology). The next day, cells were washed twice with a large volume of PBS and incubated in PLA probes (DUO92002 and DUO92004, Sigma-Aldrich) for 1 h at 37 °C. Then, the cells were washed with 1 × Duolink for two times In Situ Wash Buffer A (DUO82049, Sigma-Aldrich) and incubated with ligation mix at 37 °C for 30 min. Subsequently, the cells were washed with 1 × Duolink In situ Wash Buffer A twice and incubated with amplification mix (DUO92008, Sigma-Aldrich) at 37 °C for 100 min. Finally, the cells were washed twice with 1 × Duolink in situ wash buffer B, washed once with 0.01 × Buffer B and mounted with Duolink in situ mounting medium with DAPI (DUO82040, Sigma-Aldrich). The pictures were captured under LSM 880 confocal microscope (Zeiss, Germany).

HepG2 and Huh7 cells in 10-cm cell-culture dishes at 90% confluency were collected and lysed in 1 ml RIPA buffer (87787, Thermo Fisher Scientific) containing 1 × protease inhibitor cocktail (78438, Thermo Fisher Scientific) and 1 × phosphatase inhibitor cocktail (78426, Thermo Fisher Scientific) for 20 min on ice. The protein-containing supernatants were cleaned by centrifugation at 13,000 g for 20 min at 4 °C. 10% volume of protein lysate was kept as input control and the left lysate was mixed with 25 μl Protein A/G magnetic beads (88803, Thermo Fisher Scientific) and rotated at 4 °C for 1 h to reduce any non-specific binding. Then, the pre-cleared lysate (500–1,000 μg) was incubated with anti-FLAG antibody (F3165, Sigma-Aldrich), anti-HA antibody (51064-2-AP, Proteintech), anti-eIF3a antibody (ab86146, Abcam), anti-eIF3b antibody (sc-137214, Santa Cruz Biotechnology), normal mouse IgG (12–371, Millipore) or normal rabbit IgG (12–370, Millipore) under rotation for 1 h at 4 °C, followed by an overnight incubation with 25 μl of pre-washed Protein A/G magnetic beads under rotation at 4 °C. The proteins were collected by magnetic stand, followed by three times washing with IP washing buffer (10 mM Tris–HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Triton-X, 0.2 mM sodium orthovanadate) and then detected by Western blotting.