Camkii β

Protein levels were measured by the Bradford method. For the cross-linking experiment, 20 μg of protein from each rat was loaded in each lane of a 4–15% Tris-HCl polyacrylamide gradient gel (Bio-Rad) for electrophoresis. For analysis of the PSD fractions, 5 μg of protein was loaded in each lane of a 10% Tris-HCl polyacrylamide gel. Following transfer, membranes were incubated in blocking solution [5% non-fat milk in Tris buffered saline containing Tween 20 (TBS-T buffer)] for 1 hour at room temperature. Membranes were then incubated overnight at 4°C in primary antibody [GluA1, 1:500; GluA2, 1:5000; pGluA1(S831), 1:500; pGluA1(S845), 1:1000; TH, 1:2000; Millipore; β-actin, 1:10000; CaMKIIα, 1:5000; Syntaxin, 1:5000; Sigma-Aldrich; pCaMKIIα(T286), 1:1000; NR2b, 1:1000; PSD95, 1:2000; Stargazin, 1:1000; Cell Signaling; CaMKII β, 1:1000; Invitrogen; PKA, 1:500; AKAP150, 1:5000; Santa Cruz]. After extensive washing in TBS-T buffer, membranes were then incubated in an HRP-conjugated secondary antibody (anti-rabbit, 1:10000; anti-mouse, 1:10000; Upstate) for 60 minutes followed by further extensive washing. The protein bands were visualized using a chemiluminescence detection system (ECL Advanced). Blots were digitally imaged using the GeneSnap Bio Imaging System and quantified using Genetool software (Syngene).