RNA Binding Assay of ZFP36L2 Proteins

Protein extracts were prepared from HEK 293T cells transfected with constructs driven by the CMV promoter followed by either mZFP36L2-HA or hZFP36L2-DDK, and empty vector. Protein extracts were incubated for 15 min at room temperature with 0.2 × 10⁵ cpm of ³²P-labeled RNA probe in a final volume of 26 µL containing 10 mM HEPES (pH 7.6), 40 mM KCl, 3 mM MgCl₂, 0.5 µg/µL heparin, and 1.2 μg yeast tRNA, as previously described (Lai et al. 2000 (link); Ball et al. 2014 (link)). The resultant reaction mixtures of protein–RNA complexes were then loaded onto 6% nondenaturing acrylamide (37.5:1) gels and subjected to electrophoresis at 150 V for 15 min followed by electrophoresis at 200 V for 90 min in 0.4× Tris-borate/EDTA running buffer. The gel was dried, exposed to film (Carestream BIOMAX MR Film), and developed after 20 h of exposure. Dried gels were also exposed to a PhosphorImager screen and scanned using a Typhoon 8600 imager. ImageQuant software (GE Healthcare) was used to quantify the amount of bound and unbound probes for each EMSA. Values were normalized to blot the background for each experiment.