3002 Hongmei Fu et al. Eur. J. Immunol. 2011. 41: 2997–3005 Figure 4. Inhibition of arginase has no effect on systemic T-cell proliferation. Splenocytes from Thy1.11 Rag2 / Marilyn mice were stained with CFSE and adoptively transferred into B6 females (Thy1.21) by i.v. injection. On the same day (day 0), all recipients were immunized with male splenocytes. From day 1 to day 7, B6 female mice received intraperitoneal injections of either nor-NOHA at 100 mg/kg/day or PBS. At days 5 and 10, three mice from each group were sacriﬁced and splenocytes were analysed by two-colour ﬂow cytometry. Lymphocytes were gated on forward and side scatter and shown for expression of Thy1.1 (y axis) and CFSE (x axis). Three animals were used per time point for each group. Error bars indicate SD. Student’s t-test was used to generate the p-values described. Figure 5. Nor-NOHA does not increase NO production by MCECs exposed to a cocktail of cytokines. MCECs were treated with different concentrations of the proinﬂammatory cytokines IL-1, TNF-a and IFN-g (10–100 ng/mL) and cultured in the presence or absence of nor-NOHA (0.6 mM) or L-NAME (5 mM), for 48 h. (A) NO level in the supernatant of cultured cells using Griess reagent following conversion of nitrate into nitrite. (B) Analysis of cell apoptosis by Annexin-V staining. Data are representative of three independent experiments. Student’s t-test was used to generate the p-values described. the gene. Quantiﬁcation of arginase II mRNA was carried out using the primers 50-CTGCCATTCGAGAAGCTGG-30 (forward) and 50-GGGATCATCTTGTGG ACATTAG-30 (reverse) (spanning 104 bp) [41]. Quantiﬁcation of hypoxanthine phosphoribosyl transferase (HPRT) mRNA was carried out using the primers 50-ATGAT CCAGTCAACGGGGGAC-30 (forward) and 50-CCAGCAAGCTTG CAACCTTAACCA-30 (reverse) (spanning 177 bp). methanol. Blocked with normal goat serum (1:25 with PBS) and then treated with 0.1% Triton X-100 in PBS. After three washes in PBS, sections were incubated with either rabbit anti-mouse arginase I or rabbit anti-mouse arginase II (Santa Cruz, USA) diluted 1:100 for 18 h at 41C. After washing in PBS, sections were incubated with horseradish peroxidase- (HRP) conjugated goat anti-rabbit IgG (Dako, Denmark) diluted 1:50 for 1 h at room temperature. After washes with PBS, the chromogen 3,30-diaminobenzidine tetra- hydrochloride (DAB enhanced liquid substrate system; Sigma, D3939) was applied at 100–200 mL for 2–5 min. The sections were washed in tap water before haematoxylin counterstaining for 30 s. Western blotting Corneal tissue was ground using the beads as described above. After centrifugation, the supernatant was separated on 10% SDS- PAGE under reducing conditions and transferred to nitrocellulose membrane using electrophoresis. Membranes were probed using either rabbit anti-mouse arginase I antibody or rabbit anti-mouse arginase II antibody, followed by HRP goat anti-rabbit IgG antibody. Blots were developed using ECL plus system (Amer- sham Pharmacia Biotech). Immunoperoxidase staining Arginase activity Tissue sections (8 mm, embedded in OCT) were allowed to dry at room temperature for 2 h, treated with 3% H2O2 in The enzymatic activity of arginase was measured as previously described [42]. & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu