Anti α camkii

v-Myc-, Gag-, and Max-specific rabbit antisera were described previously [11 (link), 26 (link), 37 (link)]. The monoclonal mouse antibodies anti-α-tubulin and anti-FLAG were purchased from Sigma-Aldrich (T5168; F3165). Mouse monoclonal anti-CaM was purchased from Merck Millipore (05-173), anti-HA from Covance (MMS-101P), anti-α-CaMKII from Santa Cruz (sc-13141), and anti-pan-Ras from Thermo Fisher Scientific (MA1-012). Mouse monoclonal anti-lamin A/C (4777), rabbit monoclonal anti-c-Myc (13987), and rabbit monoclonal anti-GAPDH (5174) were obtained from Cell Signaling Technology. Monoclonal mouse antibodies specific for v-Src (327) were obtained from Calbiochem. SDS-PAGE, immunoblotting, and immunoprecipitation were carried out as described (11). For densitometry, relative protein expression levels were determined with the program ImageQuant TL (GE Healthcare).

Cerebellar tissue slices lysates of 150 μm thickness were incubated for 10 min in any of three conditions: low [K]o (1 mM), high [K]o (50 mM), or high [K]o with mibefradil (1 μM) and Ni²⁺ (300 μM). Homogenates were then made in the same solutions using a hand held glass homogenizer. Eluted lysates were loaded on 6–10% Tris-glycine gel and resolved using SDS-PAGE. Samples were transferred to 0.2 μm PVDF membrane (Millipore, Etobicoke, ON, Canada) and Western blot analysis performed using a monoclonal mouse anti-αCAMKII (1:1000; Santa Cruz, Dallas, TX, USA) or a polyclonal rabbit anti-p-αCAMKII (Thr286) (1:1000; Santa Cruz, Dallas, TX, USA). The secondary antibodies used were the appropriate mouse or rabbit antibodies conjugated to HRP (1:5000; Molecular Probes, Eugene, OR, USA) and reacted with ECL solution (Life Technologies, Burlington, ON, Canada).