Amersham ecl goat anti mouse hrp linked secondary

Protein concentration was determined using the Pierce BCA protein assay (Thermo-Fisher). Before undergoing SDS-PAGE, samples were boiled in Laemmli sample buffer for 5 min at 95°C. Proteins were separated in a resolving phase polymerized from 7%–9% acrylamide, then transferred to a polyvinylidene difluoride membrane at 24 V for 1–2 h at 4C. Membranes were blocked in 5% milk or BSA and incubated with anti-CaMKIIa (1:4000, CBa2, available at Invitrogen but made in house), pT286-CaMKII (1:2500, Phospho-Solutions), anti-GluN2B (1:1000, Cell Signaling), DAPK1 (1:800, Sigma-Aldrich), followed by either Amersham ECL goat anti-mouse HRP-linked secondary 1:10000 (GE Healthcare) or goat anti-rabbit horseradish peroxidase conjugate 1:10000 (Bio-Rad). Blots were developed using chemiluminescence (Super Signal West Femto, Thermo-Fisher), imaged using the Chemi-Imager 4400 system (Alpha-Innotech), and analyzed by densitometry (ImageJ). Variations in sample loading were corrected using b-actin (1:2000, Cell Signaling) as a loading control. Relative band intensity was normalized as a percent of control conditions on the same blot, which was set at a value of one to allow for comparison between multiple experiments.

Western analysis was performed similar as described previously (Cook et al., 2021 (link); Tullis et al., 2020 (link)). Protein concentration was determined using the Pierce BCA protein assay (Thermo-Fisher). Before undergoing SDS-PAGE, samples were boiled in Laemmli sample buffer for 5 min at 95°C. Proteins were separated in a resolving phase polymerized from 7.5% acrylamide, then transferred to a polyvinylidene difluoride membrane at 24 V for 1–2 h at 4°C. Membranes were blocked in 5% milk or BSA and incubated with anti-CaMKIIα (1:4000, CBα2, available at Invitrogen but made in house), pT286-CaMKII (1:2500, Phospho-Solutions), anti-GST (1:1000, Millipore), followed by either Amersham ECL goat anti-mouse HRP-linked secondary 1:10000 (GE Healthcare) or goat anti-rabbit horseradish peroxidase conjugate 1:10000 (Bio-Rad). Blots were developed using chemiluminescence (Super Signal West Femto, Thermo-Fisher), imaged using the Chemi-Imager 4400 system (Alpha-Innotech), and analyzed by densitometry (ImageJ). Phospho-signal was corrected to total protein. Relative band intensity was normalized as a percent of control conditions on the same blot, which was set at a value of one to allow for comparison between multiple experiments.