KCR1 Modulates If Current Figure 1. Analysis of protein interaction between HCN2 and KCR1 and expression of KCR1 in various cell types. (A) KCR1 and HCN2 can associate 3 in protein complexes. Coimmunoprecipitation of HCN2 and KCR1 from mammalian cell extracts. Protein extracts from cells transfected with pCFLAG - KCR1 (lane 0), HCN2 plus pCFLAG3-KCR1 (lanes 1), HCN2 plus p3xFLAG-CMV (lanes 2) or non-transfected cells (lanes 3) assayed by Western blot using anti-HCN2 antibody. Input: input lysates were blotted to show that HCN2 protein was successfully produced and detected. Anti-FLAG-Sepharose: 700 mg of total lysate immunoprecipitated with anti-FLAG-Sepharose shows that HCN2 and KCR1 (pCFLAG3-KCR1) can be coimmunoprecipitated (lane 1). A-Sepharose: mock immunoprecipitation using protein A-Sepharose beads unlinked to FLAG-antibody to exclude any unspecific antibody binding. (B) KCR1 message (218 bp) can be detected in all cell types tested, except for non-transfected CHO cells (upper panel). Representative electrophoresis gel illustrating results obtained in single-cell RT-PCR experiments (as described in Methods) with three different cells types. CHO: Non- transfected Chinese hamster ovary cells; CHO+KCR1: CHO cells transfected with KCR1 cDNA; RatNN: Neonatal rat cardiomyocytes; RatA: Adult rat cardiomyocytes. GAPDH mRNA (318 bp) was used as a control and is found in all cell types tested, including non-transfected, KCR1-negative CHO cells (lower panel). Negative controls did not include RNA or reverse transcriptase and gave no amplicons (not shown). (C) Regional differences in expression of KCR1 in adult rat and pig heart. KCR1 mRNA levels determined by quantitative real-time PCR (qPCR; n = 3–4). RA = right atrium, RV = right ventricular free wall, LA = left atrium, LV = left ventricular free wall, Se = septum, SAN = sinoatrial node, AV = atrioventricular node. Values are mean6SEM. *p,0.05 vs. AV node, #p,0.05 vs. SAN. Upper panel: Representative electrophoresis gel obtained from qPCR products of different cardiac regions (the AV-node region was divided in: AVS = superior part and AVI = inferior part), samples were loaded and normalized to GAPDH. doi:10.1371/journal.pone.0001511.g001 PLoS ONE | www.plosone.org 2 January 2008 | Issue 1 | e1511