Anti hakai

Cultured mESCs were rinsed briefly in PBS and then fixed and permeabilized with pre-chilled methanol:acetone (1:1, v/v) for 10 min at −20°C, or cells were fixed with PBS containing 4% paraformaldehyde for 10 min at room temperature and then permeabilized with PBS containing 0.1% Triton X-100 for 10 min. Cells were subsequently washed with PBS for three times. Cells were blocked for 30 min with 1% BSA in PBS at room temperature. Primary antibodies were diluted in blocking buffer at different concentrations (see blow) and incubated overnight at 4°C. Washed twice with PBS, cells were incubated with DAPI (dilution 1:2000, Solarbio) and fluorescent dye-conjugated secondary antibodies diluted in blocking buffer for one hour at room temperature and then visualized.
Primary antibodies concentrations: anti-Zc3h13 (1:200, Bethyl); anti-WTAP (1:200, Proteintech); anti-Virilizer (1:200, Bethyl); anti-Hakai (1:200, Bethyl); anti Mettl3 (1:200, Abcam); anti Mettl14 (1:200, Sigma-Aldrich); anti SC35 (1:500, Abcam); anti HA (1:1200, Cell Signaling, Cat#3724); anti HA (1:100, Cell Signaling, Cat#2367).

mES cells were lysed in hypotonic buffer (10 mM HEPES, pH7.5, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 1x Protease Inhibitor Cocktail (Roche) and 1 mM PMSF) on ice for 15 min, and then NP-40 was added to a final concentration of 0.25% for another 5 min. Samples were centrifuged for 3 min at 2000 rpm at 4°C, and the supernatant was saved as cytoplasmic fraction. The nuclear pellet obtained from the low speed centrifugation was washed with hypotonic buffer once and re-suspended in RIPA buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1x Protease Inhibitor Cocktail and 1 mM PMSF) and incubated on ice for 20 min. This sample was saved as nuclear fraction. SDS loading buffer was added in the samples and boiled for 10 min. The samples were loaded on 7.5% SDS-PAGE gels and subjected to immunoblotting with different antibodies. The intensity of the band was measured by Bio-Rad Image Lab software (Bio-rad).
Primary antibodies concentrations used in immunoblotting are as below: anti-Zc3h13 (1:3000, Bethyl); anti-WTAP (1:3000, Proteintech); anti-Virilizer (1:3000, Bethyl); anti-Hakai (1:3000, Bethyl); anti Mettl3 (1:3000, Abcam); anti Mettl14 (1:2000, Sigma); anti-Lamin B1 (1:5000, Proteintech); anti-α-Tubulin (1:5000, Proteintech).