Quantifying CaMKII Isoforms by Western Blot

As previously described [18] (link), tissue was homogenized using an Omni TH homogenizer, in a (1∶10 w/v) detergent- containing lysis buffer (tissue protein extraction reagent with Halt protease and phosphatase inhibitor cocktail: Thermo Scientific, Waltham, MA). Extracts were centrifuged at 12,000×g for 20 min at 4°C in a Beckman Microfuge 18, and supernatants collected for measurements. Western blots were run using 10 µg of the detergent-solubilized fraction prepared in SDS loading buffer (LI-COR Biosciences, Lincoln, NE). The sample was separated on a 10% NuPAGE Novex Bis-Tris Midi Gel (Life technologies). Proteins were transferred to nitrocellulose membrane using a dry blotting system (iBlot Life Technologies). Blots were probed using reagents and manufacturer recommendations for Odyssey Infrared imaging system (LI-COR Biosciences). Blots were probed for the following primary antibodies: mouse anti-CaMKIIα (1∶10,000, Abcam, Cambridge, United Kingdom, Cat no. ab2725); mouse anti-CaMKIIβ (1∶10,000, LifeSpan Biosciences, Cat no. LS-C21191). Mouse anti-β-actin (1∶10,000, Cell Signaling, Danvers, MA, Cat no. 37005) was used as a loading control. Blots were visualized and analyzed on the Odyssey Infrared imaging system (LI-COR Biosciences).