Modulation of Calcium-Dependent Inactivation Figure 1. Tissue-specific expression patterns of mRNAs of the main components of bAR signaling in the dLGN of Long Evans rats. RT-PCRs of mRNA from whole brain extracts (A) and VB (B) show that the a1C subunit (colored squares) as well as other Ca2+ channels forms are widely expressed. The skeletal muscle form a1S was not found in both regions (as well as in dLGN, see Figure F). In all experiments, appropriate markers for size were used in order to assure detection of specific forms of Ca2+ channels. (C) Using specific primers for all three types of bAR, we have shown specific expression of only b1 and b2AR. b3AR was neither expressed in VB nor in dLGN. (D) Following the same procedure, we have shown the expression of some of the G proteins coupled with metabotropic receptors (Ga and Gi) in dLGN. In all experiments specific markers for size were used. (E) Expression of three different adenylate cyclases, namely type 1, 6 and 8 with two isoforms A/B and C in dLGN. doi:10.1371/journal.pone.0027474.g001 the voltage step to +10 mV (upper arrowhead in Figure 3B), and A1 (Imax, Figure 3C) representing the maximal current amplitude (lower arrowhead, Figure 3B), as measured without a preceding conditioning pulse. GraphPad Prism 5.0 and Microcal Origin 6.0 software were used for data analysis and figure plotting. Drugs The following drugs were used: isoproterenol hydrochloride, propranolol hydrochloride, xamoterol hemifumarate, salmeterol, BRL 37344, sodium salt, CGP 20712 dihydrochloride, ICI 118,551 hydrochloride, SR 59230A hydrochloride, okadaic acid, PKA inhibitor myristoylated PKI 14–22 amide (all from Tocris, USA). Equimolar concentrations of ascorbic acid were added to the extracellular bath solution in order to prevent oxidation of the drugs. The selective AKAP inhibitor InCELLectTM AKAP St-Ht31 and the corresponding control peptide were purchased from Promega (Germany). Cell culture After incubation for 5 minutes at room temperature with 2 ml Trypsin solution, immortalized kidney cells of the African green monkey (COS-7 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 was done according to the manufacturer’s instructions (Invitrogen, Germany). DNA-Lipofecta- mine complexes were added into the flask and cells were left in incubator for the next two days. 24 hours after transfection sodium- butyrate was added in order to enhance expression of transfected proteins. 48 hours after transfection, the cells were lysed and obtained lysates were either used immediately or stored at 220uC. For GFP constructs, transfection efficiency was analyzed under a fluorescence microscope (Zeiss axioplan microscope, Carl Zeiss, Jena, Germany). PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e27474