Tc dapk 6

The IRE1 RNase inhibitor 4μ8c was purchased from EMD Millipore (catalog no. 412512), ER Stress Inducer Tunicamycin was purchased from Tocris (catalog no. 3516), potent and selective, ATP-competitive inhibitor of death-associated protein kinase 1 (DAPK1) TC-DAPK 6 (catalog no. 4301). Pin1 inhibitor Sulfopin (PIN1-3), which is a highly selective covalent inhibitor of Pin1was purchased from Selleckchem (Catalog No.S9782).

For imaging experiments, neurons grown on coverslips were treated every day from either DIV14 or DIV18 until DIV20. Ifenprodil hemitartrate (Tocris Bioscience; Cat# 0545) and memantine hydrochloride (Tocris Bioscience; Cat# 0773) were dissolved in sterile H₂O and used at a final treatment concentration of 3 μM. The DAPK1 inhibitor TC-DAPK6 (DKI; Tocris Bioscience; Cat# 4301) was dissolved in DMSO and used at a final concentration of 1 μM (0.01% DMSO). Cultures were fixed and immunostained at DIV21.
For biochemistry, culture media was replaced with conditioned media containing 10 μM DKI or DMSO (0.1%) for 1 h. Cells were collected by scraping immediately following treatment, centrifuged, and frozen at −20°C for later processing. Pellets were lysed by pipetting in gentle lysis buffer to solubilize only non-synaptic proteins. Samples underwent SDS–PAGE and Western blotting as described above for the detection of protein and phosphorylation levels.