Wt islets (∼1,000) were lysed in co-IP lysis buffer containing 20 mM Tris ·HCl (pH 8.0), 137 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, and a mixture of protease and phosphatase inhibitors (Roche). After centrifugation (16,000g at 4 °C for 10 min), the supernatant was divided into two aliquots. Immunoprecipitation studies were performed as described68 (link). In brief, samples were incubated overnight at 4 °C with either an anti-CaMKIIδ antibody (goat IgG, Santa Cruz) or the same amount of goat IgG (Santa Cruz). After this step, reaction mixtures were incubated with protein A/G PLUS agarose beads (Santa Cruz) for 4 h at 4 °C. The immunoprecipitated proteins were washed three times with a buffer containing 10 mM Tris ·HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100, followed by SDS-PAGE analysis.
Immunoblots were probed with anti-barr2, anti-CaMKII (pan), and anti-CaV1.2 (α1 (α1 subunit69 (link)) antibodies, using the Quick Western Kit (IRDye 680RD, LI-COR Biosciences). Western blots were scanned and validated with an infrared imaging system (Odyssey CLx; LI-COR Biosciences). The antibodies used are listed in theSupplementary Table 2 above.
Immunoblots were probed with anti-barr2, anti-CaMKII (pan), and anti-CaV1.2 (α1 (α1 subunit69 (link)) antibodies, using the Quick Western Kit (IRDye 680RD, LI-COR Biosciences). Western blots were scanned and validated with an infrared imaging system (Odyssey CLx; LI-COR Biosciences). The antibodies used are listed in the