Quantifying EDC4 Puncta in Transfected Cells

Transfected cells were washed once with PBS then fixed in 4% paraformaldehyde for 30 min. Cells were blocked and permeabilized in 10% normal goat serum and 0.1% Triton-X100 in PBS for 1 h. Cells were then stained with a knockout-validated antibody against enhancer of mRNA decapping 4 (EDC4) (1:200, Abcam) (Hallacli et al., 2022 (link)) at room temperature for 1 h before washing three times with PBS. Secondary antibody (Invitrogen) was then applied for 1 h, slides washed three times with PBS, and incubated with DAPI solution (Invitrogen) to stain nuclei. Slides were coverslipped with Prolong Glass mounting media (Invitrogen) and stored at -20°C until imaged using a Zeiss LSM800 confocal microscope. ImageJ was used to automate EDC4⁺ puncta counts in GFP⁺ cells to remove bias.