Sc 25743

Tissue samples of control and pilocarpine-treated animals containing only the subiculum were homogenized at 4°C in extraction buffer [150 mM NaCl, 50 mM Tris, 10 mM HEPES, 1% Triton X-100, 1% EGEPAL, proteinase inhibitor cocktail (Roche)]. Homogenates were centrifuged at 20,000 g for 15 min and the resulting supernatant was used for SDS-PAGE and protein determinations with the BCA assay (Pierce, Rockford, IL, USA). Western blot analysis was performed using 5–20% gradient SDS-PAGEs. Gels were electro blotted on PVDF membranes (Roth, Karlsruhe, Germany) for 2 h and membranes subsequently blocked for 1 h in TBST buffer (100 mM Tris-HCl; 0.9% NaCl, 1% Tween 20, pH 7.4) containing 5% non-fat dry milk. Blots were incubated overnight at 4°C with specific primary antibodies (anti AC-1 1:200, SC-25743, Santa Cruz Biotechnology, Santa Cruz, CA, USA, anti-Actin 1:5,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and immunoreactivity was visualized using HRP-coupled goat anti-rabbit or goat anti-mouse secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Quantification of Western blots was done by densitometric analysis using the NIH Image program v1.61. The relative expression levels of adenylyl cyclase 1 (AC1) and actin as well as the ratio of AC1 and actin are expressed as mean values ± standard error of mean (SEM).

The cells were lysed in a RIPA lysis buffer. The supernatant was collected, and the protein concentration was calculated with a BCA protein assay kit (Thermo Scientific, USA). ADCY1, EPAC2, and GAPDH antibodies were purchased from Santa Cruz Biotechnology (sc-25743, sc-28326, and sc-32233, respectively). CD63, CD81, CD9, and TSG101 antibodies were purchased from Abcam (Cambridgeshire, UK, ab21735, ab109201, ab92726, and ab125011, respectively).