Anti phospho camkii

The cardiac tissue cells were rinsed and homogenized in RIPA lysis buffer containing protease inhibitor PMSF. The insoluble protein lysate was removed by centrifugation at 12,000 rpm for 5 min at 4°C. 50 μg of the protein lysate was resolved using 12% SDS–polyacrylamide gel electrophoresis. The gels were transferred to polyvinylidene difluoride (PVDF) membranes by semidry electrophoretic transfer at 200 mA for 60 min. The PVDF membranes were blocked one hour in 5% milk at room temperature and subjected to Western blot analysis. Following antibodies were used: anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-PKB (Ser473), anti-phospho-CaMKII, and anti-PKB (Cell Signaling Technology, Beverly, MA, USA), anti-ERK1/2, anti-GAPDH and anti-eIF-5 (Santa Cruz Technology, Delaware, CA, USA). The sheets were analyzed with antibodies according to the supplier’s protocol, and visualized with peroxidase and an enhanced-chemiluminescence system (ECL kit, Pierce Biotechnology, Inc.). Bands were visualized by use of a super western sensitivity chemiluminescence detection system (Pierce, IL). Autoradiographs were quantitated by densitometry (Science Imaging system, Bio-Rad, Hercules, CA), and the ratio was compared between the phosphorylated p-ERK1/2, p-AKT, p-CaMKII and total proteins ERK1/2, AKT, CaMKII, respectively.

Brain tissues were homogenized in lysis buffer (150 mM NaCl, 10 mM NaH₂PO₄, 1 mM EDTA, 1% Triton X-100, 0.5% SDS) with protease inhibitor cocktail and phosphatase inhibitor (Sigma). Equivalent amounts of protein were analyzed by 4–20% Tris-Glycine gel electrophoresis (Invitrogen). Proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies. Visualization was enabled using enhanced chemiluminescence (GE Healthcare Pharmacia). The following primary antibodies (dilutions) were used: anti-phosphoCREB (1:1000, Cell Signaling, #9198), anti-CREB (1:1000, Cell Signaling, #9197), anti-phosphoERK (1:1000, Cell Signaling, #9106), anti-ERK (1:1000, Cell Signaling, #9102), anti-phospho-CaMKII (1:1000, Cell Signaling, #12716), anti-CaMKII (1:1000, Cell Signaling, #4436), anti-PSD95 (1:1000, Cell Signaling, #36233), anti-BDNF (1:1000, Sigma, #AB1534SP), Iba-1 (1:1000, Santa Cruz Biotechnology, sc-32725), CD68 (1:1000, Bio-Rad, MCA341GA), Dectin-1 (1:1000, Invitrogen, PA5-34382), CD11b (1:1000, Bio-Rad, MCA711), and β-actin (1:5000, Cell Signaling, #3700). Secondary antibodies were HRP-conjugated anti-rabbit or anti-mouse antibodies (1:1000, Cell Signaling, #7074 and #7076).