J Physiol 587.1 SNAT4 function in human placenta 63 endogenous perxoidase activity with 3% aqueous H2O2, followed by a 30 min incubation with non-immune block (10% normal swine serum, 2% normal human serum (‘in-house’), 0.1% Tween-20 in TBS). Primary anti- bodies (afﬁnity-puriﬁed rabbit IgG) were custom-made (Eurogentec: Seraing, Belgium) using the following amino acid sequences of the human system A isoforms as anti- gen: SNAT1 – VPEDDNISNDSNDFT (residues 18–32); SNAT2 – SNLGKKKYETEFHPG (residues 55–69); SNAT4 – YGEVEDELLHAYSKV (residues 391–405). Antibodies were diluted to 1 μg ml−1 in non-immune block and applied to sections overnight at 4◦C. For negative controls, and to ensure antibody speciﬁcity, primary antibodies were preadsorbed with the appropriate puriﬁed antigenic peptide (10× excess) overnight at 4◦C before applying to serial sections at 1 μg ml−1. Following repeated washes with TBS containing 0.6% Tween-20, antibody binding was detected by sequential application of biotinylated swine anti-rabbit IgG (Dako: Ely, UK; 1 : 200 dilution) and avidin-peroxidase (20 ng ml−1), with the chromogen diaminobenezidine, to produce a brown precipitate. Sections were counterstained with Harris’ haematoxylin, dehydrated and mounted with DPX. Preparation of MVM vesicles MVM vesicles were prepared using the method of Mg2+ precipitation and differential centrifugation as previously described (Glazier et al. 1988; Mahendran et al. 1994) with the modiﬁcation that Mg2+ concentration was increased to 12 mM for ﬁrst trimester MVM. Once isolated, MVM vesicles were suspended in intravesicular buffer (IVB: 30 mM KCl, 110 mM choline chloride, 20 mM mannitol, 6.5 mM Tris, and 13.5 mM Hepes, pH 7.4). MVM protein concentration was determined by the method of Lowry et al. (1951). Vesicle purity was assessed by measuring enrichment of alkaline phosphatase activity as previously described (Glazier et al. 1988; Mahendran et al. 1994). Alkaline phosphatase enrichment factors (mean ± S.E.M.) for ﬁrst trimester and term MVM vesicles (n = 6 for both) were 18 ± 2 and 19 ± 2, respectively. Statistical analysis revealed no signiﬁcant difference between the two groups (P = 0.82, Mann–Whitney U -test). All vesicles intended for activity experiments were stored at 4◦C prior to measurement of amino acid uptake, performed within 48 h of isolation. MVM vesicles intended for Western Blot analysis were stored at −80◦C. Western blot analysis In order to compare SNAT4 protein expression in MVM from ﬁrst trimester and term placenta, 30 μg protein was mixed at a ratio of 1 : 1 with loading buffer (22% glycerol, pH 6.8, 154 mM SDS, 4.4 M urea, 0.002% C 2009 The Authors. Journal compilation C 2009 The Physiological Society bromophenol blue, and 10% v/v 2-mercaptoethanol, 139 mM Tris-HCl) and heat reduced for 5 min at 95◦C. BeWo choriocarcinoma cell homogenate was included as a negative control for SNAT4 expression as shown previously (Desforges et al. 2006) in order to conﬁrm primary antibody speciﬁcity. Polyacrylamide gel electro- phoresis was performed using 3% stacking gel and 10% resolving gel. Proteins were then electrotransferred onto nitrocellulose membrane. The membranes were blocked for 1 h at room temperature with 5% dried skimmed milk powder in phosphate-buffered saline (PBS; 172 mM NaCl, 3.35 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and then washed for 3 × 5 min in PBS–0.1% Tween-20 (PBS-T). A rabbit polyclonal to SNAT4 (Abcam: Cambridge, UK) was used at a 1 : 300 dilution in PBS-T. Primary and HRP-conjugated goat anti-rabbit secondary antibody (Dako, Ely, UK; 1 : 2000 dilution in PBS-T) incubations were carried out for 1 h at room temperature. Immunoreactive signals were detected by enhanced chemiluminescence (ECL). Following ECL, the nitrocellulose membrane was reprobed for β-actin (mouse anti-β-actin monoclonal, 1 : 1000 dilution) to conﬁrm equal protein loading. Measurement of total system A activity in MVM vesicles MVM vesicles from ﬁrst trimester and term placenta were used at 5 μg ml−1 and 10 μg ml−1, respectively (diluted in IVB). MVM vesicles were then incubated in 4 μm valinomycin, a potassium ionophore, for 1 h at room temperature as previously described (Ayuk et al. 2000) before transport assays were performed. [14C]MeAIB (0.4 mM, Perkin Elmer) uptake was measured at room temperature (21–24◦C) following addition of 20 μl MVM vesicles to 100 μl extravesicular buffer (EVB: 30 mM KCl, 110 mM NaCl, 20 mM mannitol, 14 mM Tris, 6 mM Hepes, pH 8.5) by the method of rapid ﬁltration (Mahendran et al. 1993, 1994; Ayuk et al. 2000). Experimental conditions were modiﬁed to promote system A activity; EVB composition achieved an inwardly directed Na+ gradient of ∼90 mM across MVM plasma membranes creating an optimal driving force for MeAIB transport and an alkali pH of 8.5 was used to stimulate SNAT activity (Hatanaka et al. 2001). [14C]MeAIB uptake in the presence of 30 mM MeAIB was also measured and considered to be non-system A mediated because at this high concentration of substrate it is predicted that all three SNAT isoforms would be saturated. The difference between total [14C]MeAIB uptake and uptake in the presence of 30 mM MeAIB was calculated to give a measure of system A-speciﬁc uptake. Previous studies have shown that [14C]MeAIB uptake by MVM after 30 s provides a reasonable estimate of initial rate (Mahendran et al.