Immunoblotting Analysis of CaMKII and GST

For immunoblot analysis, protein was transfered onto PVDF membrane (Goodell et al., 2017 (link)) and detected using antibodies specific for CaMKIIα (CBα2) or GST. Briefly, 10 μg of total protein was subjected to SDS-PAGE on 7.5% polyacrylamide gels made in house and transferred to PVDF membrane for 1–2 hrs at 4°C. In GluN2B binding assays, GST was used to assess total protein and bound CaMKII levels were determined by the ratio of bound to total CaMKII. Blots were developed using chemiluminescence (Super Signal West Femto,Thermo-Fisher) and imaged using the Chemi-Imager 4400 system (Alpha-Innotech). Densitometry was calculated in AlphaEaseFC (Alpha-Innotech) (Barcomb et al., 2014 (link); Goodell et al., 2014 (link), 2016 (link)). The relative immuno-detection value (IDV) was normalized as a percent of the average of WT conditions for the same blot, which was set at a value of one to allow comparison between multiple experiments. Sample size is indicated by individual data points on graph and statistical analysis is detailed in figure legends.