Ernight at 4 C with five g of mouse monoclonal antiFLAG (Kodak, USA). Antibodyproteins complexes have been recovered with 50 l protein G-coupled dynabeads (Invitrogen, USA) based on manufacturer’s guidelines. Soon after three consecutive washes in PBS buffer containing Ca2+ and Mg2+ the samples have been eluted by heating to 80 C for 10 min in LDS sample buffer (Invitrogen, USA) and subjected to SDS-PAGE and Western blot analysis. For co-immunoprecipitation assays with full length G13 and truncated forms of ZO-1, three.five g of a pDisplay-HA-G13 construct was co-transfected into HEK 293 cells plated on 60 mm dishes applying Lipofectamine LTX (Invitrogen, USA) with each other with three.five g of either pDipslay or various truncated forms of ZO-1, Veli-2, or PSD95 into pDisplay-FLAG. Forty-eight hours later cells were lysed on ice in 600 l lysis buffer containing 25 mM Hepes, pH 7.5, five mM MgCl2 , four mM EDTA, 1 Triton X-100, and Full protease inhibitor cocktail (Roche, Switzerland). Protein extracts had been treated essentially as described above except that 8 g 12CA5 mouse monoclonal anti-HA antibody (Roche, Switzerland) had been used for immunoprecipitation. For Western blotting, IP products or total protein lysates (30 g) had been generally separated on a denaturing 42 Bis-Tris Web page gel (Invitrogen, USA), transferred onto a hybond-P, PVDF membrane (GE Healthcare, USA) and incubated overnight at 4 C together with the proper main antibody. Mouse monoclonalanti-HA (1400; Roche, USA) or anti-FLAG (11000; Kodak, USA) or rabbit polyclonal anti-Ezrin H-276 (1500; Santa Cruz, USA) or mouse monoclonal anti-myc tag 9B11 (11000; Cell Signaling Technologies, USA). The membrane was subsequently processed employing the SNAP id system (Millipore, USA) and signal was detected with an Dynorphin A (1-8) Biological Activity HRP-coupled secondary antibody in addition to a chemiluminescent substrate (Supersignal West Pico, Pierce, USA) on a Chemidoc imager (Biorad, USA). Quantification and normalization was performed utilizing ImageLab (Biorad, USA). When vital membranes have been stripped making use of a stripping option (Uptima, USA) and reprobed with an additional primary antibody. To analyze the expression in the PDZ domain-containing proteins and test the specificity in the antibodies applied for immunohistochemistry circumvallate papillae and whole olfactory epithelia of fifteen six weeks old C57BI6J mice had been collected and pooled with each other. Tissue lysates have been prepared in lysis buffer utilizing a tissue lyser (Qiagen, Germany) for the duration of three cycles of 90 s each and every at 20 Hz. After centrifugation the soluble fraction was recovered and the protein content material assessed. Seventy-five microgram of each and every lysate were separated on denaturing 42 Bis-Tris Web page gel (Invitrogen, USA), transferred onto a hybond-P, PVDF membrane (GE Healthcare, USA) and incubated overnight at four C together with the proper main antibody. Mouse monoclonal anti-actin (11000; A5441; Sigma, USA), or rabbit polyclonal antiGOPC (1500; SAB3500332, Sigma, USA), or rabbit polyclonal anti-ZO-1 (1600; 40200; Invitrogen, USA), or mouse monoclonal anti-MPDZ (1250; 611558; BD Tranduction Laboratories, USA), or goat polyclonal anti-G13 (1200; sc-26781; Santa Cruz Biotechnology, USA). The membrane was subsequently processed as described above. Comparison in the expression levels of ZO-1 and G13 in postnatal and adult mice was carried out by collecting olfactory epithelia from 6 P0, 3 P30, and 15 adult animals, pooling the samples in the animals of the same age and preparing tissue lysates as described above. 75, 100, and 130 g o.