Camk2

Single‐molecule fluorescence in situ hybridization for miRNA detection on hippocampal cultures was performed using the QuantiGene ViewRNA miRNA Cell Assay Kit (Thermo Fisher) according to the manufacturer's protocol with slight modifications. To preserve dendrite morphology, protease treatment was reduced to a dilution of 1:10,000 in PBS for 45 s. The probes hsa‐miR‐499a‐5p (Alexa Fluor 546; Thermo Fisher) and CamK2 (Alexa Fluor 488; Thermo Fisher) were used for smFISH.

After washing with cold phosphate-buffered saline (PBS), tissues and cells were homogenized with a mixture of pH 7.4 RIPA buffer (1% Triton X-100, 10 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA) and protease inhibitor, and centrifuged at 8,000 rpm for 5 min at 4°C. A bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, Waltham, MA, United States) was used to measure the extracted proteins. Proteins were electrophoresed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose (NC) membranes. NC membranes were blocked with 5% milk dissolved in Tris-buffered saline (TBS) for 30 min. They were then incubated with primary antibodies against DAPK1 (Cell Signaling Technology, 1:1,000), DM1A (Abcam, 1:2,000), caspase-3 (Cell Signaling Technology, 1:1,000), cleaved caspase-3 (Cell Signaling Technology, 1:500), p35/25 (Cell Signaling Technology, 1:1,000), ERK1/2 (Cell Signaling Technology, 1:1,000), p-ERK1/2 (Cell Signaling Technology, 1:1,000), CaMK2 (Thermo Fisher Scientific, 1:1,000), and p-CaMK2 (Thermo Fisher Scientific, 1:1,000) at 4°C overnight. This was then followed by incubation with anti-mouse or anti-rabbit IgG conjugated to IRDye^® 800 CW (Li-Cor Bioscience, Lincoln, NE, United States, 1:10,000) at 37°C for 1 h. NC membranes were visualized using the Odyssey Infrared Imaging System (Li-Cor Bioscience, United States).