24-well plate, containing defatted, baked, and poly-D-lysine- coated coverslips. The cell cultures were incubated at 37uC and 5% CO2 up to the appropriate time points. Between 3rd and 4th day of incubation, AraC (final concentration: 6 mM) was added to prevent further growth of glial cells. Primary hippocampal cells were prepared in the same way as described for thalamic neurons and grown in culture until day 10 to 14. Some of the cover slips were taken out and immediately fixed with 4% paraformaldehyde (PFA), stained with PKARIIb antibody (1:500, BD Bioscience; see below) and used as control. For comparison cells were treated with bAR agonist isoproterenol hydrochloride (50 mM) for 5 minutes before fixation and PKARIIb antibody staining. All cells were then incubated with the appropriate secondary antibody, mounted on microscope slides, and analysed under the microscope. Finally, distances (in pixels) between the dendritic localizations of PKA and the centre of the soma were quantified using MetaMorph software (Visitron Systems GmbH, Puchheim, Germany). Immunocytochemistry After 10–14 days in vitro (DIV 10–14), PFA-fixed thalamic cells were washed two times with 25 mM glycine in phosphate buffered saline (PBS, 10 mM), one time with PBS, and subsequently preincubated at room temperature in PBS-blocking solution containing 10% horse normal serum, 2% bovine serum albumin, 5% sucrose, and 0.3% Triton X-100. After 1 h, the following primary antibodies were added in different combinations to the blocking solution and incubated for 90 min at room temperature: rabbit anti-CaV1.2 (1:200, Alomone Labs, Israel); mouse anti- cAMP-dependent protein kinase type II beta regulatory subunit (PKARIIb, 1:500, BD Bioscience, USA); rabbit anti-b2-AR (H-73 1:400, Santa Cruz, USA); mouse anti-microtubule-associated protein 2 (MAP2, HM-2, 1:1000, Sigma, Germany); goat anti- AKAP-150 (N-19, Santa Cruz, USA); sheep anti-protein phos- phatase 2A (PP2A, Acris, Germany). Cultures were then treated with PBS including 0.3% Triton X-100, incubated with appropriate specific fluorescent secondary antibodies: Alexa 488 donkey anti-sheep IgG; Alexa 488 goat anti-mouse IgG; Alexa 488 goat anti-rabbit IgG; Alexa 488 rabbit anti-goat IgG; Alexa 647 donkey anti-mouse IgG; Alexa 647 donkey anti-goat IgG; Alexa 647 donkey anti-rabbit IgG, (all from Molecular Probes, Invitrogen, Germany); Cy3-conjugated goat anti-rabbit IgG; Cy3-conjugated goat anti-mouse IgG; Cy3-conjugated rabbit anti-goat IgG, (all from Dianova, Germany) for 90 min, washed, and mounted with Mowiol. Omission of primary and secondary antibodies resulted in lack of fluorescent signals. Immunohistochemistry Long–Evans rats P25–27 were deeply anesthetized using pentobarbital (50 mg/kg body weight) and transcardially perfused with PBS, followed by an ice-cold 4% PFA/PBS for 35–40 min. Brains were removed, postfixed for 4 h in 4% PFA/PBS, and cryoprotected with 30% sucrose. Coronal sections (20 mm) were cut at the level of the dLGN, mounted onto polylysineslide glass (Menzel, Germany), and air dried. For detection of CaV1.2, fresh- frozen sections were used. In this case, brains from isoflurane- anesthetized rats were removed and frozen in 250uC isopentane. Cryostat coronal sections of 20 mm thickness were cut at the level of the dLGN, thaw-mounted onto Polylysine slide glass, air dried, and fixed in 4% PFA/PBS for 10 min. After permeabilization with 0.1% Triton X-100 in PBS for 10 min and several washeswith PBS, sections were blocked with 10% normal horse serum (NHS), 2% BSA in PBS for 3 h to minimize nonspecific binding before incubation of slices with primary antibodies (rabbit anti-CaV1.2, Modulation of Calcium-Dependent Inactivation 1:200; mouse anti-PKARIIb, 1:500) in 2% NHS, 2% BSA in PBS at 4uC for 16–18 h. After washing, sections were incubated withto Cy3-conjugated donkey anti- rabbit IgG (1:400 in 2% NHS, 2% BSA in PBS, Dianova, Germany) or Alexa488 goat anti-mouse IgG (1:750 in 2% NHS, 2% BSA in PBS, Molecular probes, Invitrogen, Germany) for 1.5 h, washed again, and mounted with Immumount. Culturing of COS-7 cells and pull down assays Dulbecco’s modified Eagle medium (DMEM+; Gibco, Egges- tain, Germany) and washing buffer PBS were warmed to 37uC and frozen trypsin aliquots were thawed at room temperature. COS-7 cells (grown to confluency) were washed once with PBS. After incubation for 5 minutes at room temperature with trypsin (2 ml), cells were collected with 10 ml cell culture medium and transferred into a 15 ml Falcon tube. Cell pellets obtained after centrifugation at 500 x g for 5 minutes at 4uC were resuspended in 10 ml of Dulbecco’s modified Eagle medium (DMEM; Gibco, Eggestain, Germany). Transfection of the cells was done according to the manufacturer’s instruction (Invitrogen, Germany). Trans- fection was conducted for 2 days and after 24 hours sodium- butyrate was added in order to enhance expression of transfected proteins. In the following cells were lysed and lysates were used immediately or stored at 220uC. For GFP constructs, transfection efficiency was analyzed under a fluorescence microscope (Zeiss Axioplan microscope, Carl Zeiss, Jena, Germany). Transfected COS-7 cells expressing proteins of interest tagged to GFP or c-myc were scraped in medium and transferred to a 15 ml Falcon tube. Cell flask was washed with 2 ml of Tris- buffered saline (TBS) and the solution was pulled with scraped cells. Content of the cells was made accessible by repeating two times centrifugation (1000 x g, 5 min, 4uC), discarding the supernatant, resuspension of cells in 1 ml of TBS, and transfer to a 1.5 ml Eppendorf tube. In order to disrupt cells, the pellet was frozen and thawed using liquid nitrogen. After careful resuspen- sion in 200 ml of TBS/Triton X-100 and vortexing, the lysate was rotated for 1 h at 4uC. A cleared lysate which was obtained after 20 min centrifugation (14000 rpm, at 4uC) was diluted with TBS containing 0.2% Triton X-100 and either used directly for pull down assay or stored at 220uC for further use. Next, 50 ml matrix beads coupled to one of the proposed binding partners with GST or MBP tag were washed 2x and equilibrated in TBS containing 0.1% Triton X-100. Appropriate lysates from COS-7 cells (200–300 ml) were added and beads were gently shaken overnight at 4uC. On the next day matrixes were centrifuged (5 min, 600 x g. 4uC) washed three times (TBS/0.2% Triton X-100, 10 min) to remove unbound proteins. Bound proteins were eluted by 5 min boiling with 4 x SDS sample buffer and either loaded on SDS-PAGE gels directly or stored at 220uC for further use. Immunoprecipitation AKAP5-GFP, co-expressed with PKARIIb-c-myc in COS-7 cells, was immunoprecipitated using mMACSTM Epitope Tag Protein Isolation Kit (Miltenyi Biotec, Germany) according to manufacturer’s instructions. Eluted probes were loaded on SDS- PAGE and presence of proteins interacting with AKAP5 was detected after Western blot analysis using a PKARIIb specific antibody (BD Bioscience, USA). Western blotting procedures For immuno-blot experiments solubilized protein fractions were separated on 5–20% SDS-polyacrylamide gradient gels and subsequently transferred to nitrocellulose membranes (90 min, PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e27474