Immunofluorescent staining of METTL17 using anti-METTL17 antibody (red) and anti-Beta Tubulin antibody (green). METTL17 was detected in immunocytochemical section of HELA cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 ug/ml rabbit anti-METTL17 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. Cy3 Conjugated Goat Anti-Rabbit IgG and DyLight 488 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of METTL17 using anti-METTL17 antibody. Lane 1: human U20S whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: human Jurkat whole cell lysates, After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-METTL17 antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. METTL17 (~51 kDa predicted) was detected at ~68-70 kDa, consistent with slower electrophoretic migration due to its mitochondrial targeting sequence and compact methyltransferase domain.