Novel CAR Exon 8 Interactions Figure 2. Human CAREx8 localization and adenovirus-mediated gene transfer is similar to hCAREx7 in cell monolayers but distinct in polarized cells. COS-7 cells transfected with hCAREx7 (A) or hCAREx8 (B) show a similar distribution. Panel C shows that CHO cells transfected with hCAREx7 or hCAREx8 mediate similar adenovirus gene transfer. Immunocytochemistry for endogenous hCAREx7 (D) or hCAREx8 (E) (green) reveals distinct localization in polarized primary human airway epithelia greater than 2 weeks of age. hCAREx7 localizes to the basolateral membrane and shows co-localization with the basolateral portion of ZO-1 (red, D, E, F). hCAREx8 localizes diffusely in the upper region of the cytoplasm with some apical staining (see arrowhead). Panel F shows ZO-1 (red) and a lack of staining with control rabbit pre-immune serum (green). Panel G shows the abundance of CAREx7 or CAREx8 transcripts in primary cultures (in vitro) or from lung tissue (in vivo). Panel H shows that the relative enrichment of CAREx7 to CAREx8 transcripts is similar in vitro and in vivo. Localization of hCAREx7 (I) or hCAREx8 (J, K, L, M) after over-expression in primary cultures and co-stained (red) for ZO-1 (I, J), acetylated a-tubulin (K), CD55/decay accelerating factor (DAF, L), or ezrin (M). Panel N shows that expression of exogenous hCAREx8 in polarized human airway epithelia mediates two-fold greater adenovirus gene transfer than hCAREx7 in comparison to control GFP transduced cells. *p,0.01. Confocal microscopy (60x oil immersion). doi:10.1371/journal.pone.0009909.g002 airway epithelia, greater than two weeks of age, were stained for endogenous hCAREx7 (Figure 2D, green), hCAREx8 (Figure 2E, green) with antibodies raised to peptides composed of the last 13 amino acids of either hCAREx7 or hCAREx8, or pre-immune serum control (Figure 2F, green), and were co-stained for the tight junction protein ZO-1 (red). Distinct patterns of localization were observed for these two isoforms. As previously shown, hCAREx7 localizes to the tight and adherens junctions of airway epithelia [23]. In contrast, hCAREx8 localizes primarily to the upper region of the cytoplasm and apical surface above ZO-1 (Figure 2E, arrow), without ZO-1 tight junction overlap. In contrast to ZO-1, co-localization was observed when co-stained with the apical protein ezrin (Figure S1). To determine the relative abundance of the two isoforms in polarized airway epithelia (in vitro) or lung tissue (in vivo), RNA was extracted from human airway epithelia greater than two weeks of age or total lung tissue, respectively, and subjected to quantitative RT-PCR (see Figure S2 for primer specificity). Despite donor variability, hCAREx8 levels were consistently markedly lower than hCAREx7 both in cultures and tissues (Figure 2G, Figure S2) with a similar ratio of hCAREx7 to hCAREx8 both in vitro and in vivo (Figure 2H). To confirm the distinct localization of these isoforms, airway epithelia, greater than two weeks of age, were transduced from the basolateral side with adenovirus containing the cDNA for hCAREx7 or hCAREx8, and subjected to immunocytochemistry 36 hours later. Recombi- nant expression levels are markedly higher than endogenous levels, thus confocal microscopy settings are set at a level that does not detect endogenous. Nevertheless, the localization of recombinant hCAR was similar to that seen with endogenous isoforms. Whereas the majority of hCAREx7 localized to the basolateral membrane (Figure 2I), hCAREx8 was largely diffusely distributed throughout the cell but was also present at the apical membrane where it appeared above ZO-1 (Figure 2J), below the cilia, marked by acetylated a-tubulin (Figure 2K), and overlapped at the same PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9909