Hpa020352

Far-Western blotting was performed by referring to a published literature^{51 (link)} with minor modifications. The eIF3 protein complex (30 μg) in rabbit reticulocytes (P1318, BioVision) was separated by a 10% SDS-PAGE and transferred to PVDF membrane. An equal amount of BSA was also loaded as a negative control. The gel was first stained with coomassie blue to ensure the proteins had been successfully transferred and then washed by wash buffer (5% ethanol and 10% acetic acid in ddH₂O) to remove the dye. Afterward, the proteins on the membrane were denatured and renatured in AC buffer (100 mM NaCl, 20 mM Tris-HCl pH 7.6, 0.5 mM EDTA, 10% glycerol, 0.1% Tween-20, 2% non-fat milk and 1 mM DTT) by gradually reducing the guanidine-HCl concentration. After blocking the membrane with 5% non-fat milk in 1 × PBST buffer at RT for 1 h, the membrane was incubated with 1 μg/ml recombinant METTL16 protein (81085, Active Motif) at 4°C overnight. After that, the membrane was washed with 1 × PBST buffer to remove the unbound protein and incubated with anti-METTL16 antibody (1:1000, HPA020352, Millipore Sigma) in the PBST buffer containing 3% milk at RT for 1h. Finally, the HRP conjugated goat anti-rabbit secondary antibody (1:5000, ab6721, Abcam) was incubated for another 1h and chemiluminescent detection was performed.

The cells were seeded onto the sterile slides and placed in the 6 well plates with the cell density of 1 × 10⁵ cells per well. After incubation for 24h, the cells were gently rinsed by phosphate-buffered saline (PBS) 3 times followed by fixed with 4% paraformaldehyde for 15min. Then, the cells were washed with PBS three times followed by permeabilized by 0.1% Triton X-100 in PBS for 15 min. To block the nonspecific binding, the cells were incubated with 2% bovine serum albumin (BSA) in PBS for 1h. The cells were subsequently incubated with primary antibodies against METTL16 (1:100, HPA020352, Sigma-Aldrich), METTL3 (1:100, ab195352, Abcam), METTL 14 (1:100, HPA038002, Sigma-Aldrich) and SC35 (1:500, ab11826, Abcam) overnight at 4°C, respectively. After washing with PBS three times, the corresponding fluorescence-labeled secondary antibodies (1:200, goat anti-rabbit IgG(H+L) Alexa Fluor 555: 4413S, Cell Signaling Technology; 1:200, goat anti-mouse IgG(H+L) Alexa Fluor 488: 4408S, Cell Signaling Technology) were applied to stain the cells and kept incubating for 1h. The nuclei were counterstained by mounting the cells in DAPI (F6057, Sigma-Aldrich). The immunofluorescent signals were photographed by a confocal laser scanning microscope (CLSM) (Carl Zeiss LSM 880).