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Supporting information Figure 1 Serum and peritoneal half life of lactoferrins following i.p. injection Mice (n = 4 per treatment group) received a single dose of 1% NLF or rice rLF by i.p. injection (total of 2.5 mg LF administered). At various times thereafter, mice were terminated, the peritoneal cavity washed with 5 ml PBS and the mice were subsequently bled by cardiac puncture. Peritoneal wash (A) and serum samples (B) were analyzed for LF content by LF-specific capture ELISA. Data are displayed as the mean (+SE) of LF concentration (log10) for NLF and rice rLF. There were no statistically significant differences in LF concentration at any time point.

Supporting information Figure 2 Digestion of NLF by B-galactosidase MALDI-TOF mass spectrometry of permethylated N-glycans from NLF after β-galactosidase digestion (A, 1500-2334 m/z, B, 2333-3167 m/z and C, 3166-4250 m/z). N-glycomic profiles were obtained from the 50% MeCN fraction from a C18 Sep-Pak column. Schematic structures are according to the Consortium for Functional Glycomics guidelines. All molecular ions are [M +Na]+. Putative structures are based on composition, tandem MS, and biosynthetic knowledge. Bracketed structures have not been unequivocally defined. Data shown are a summation of spectra from 5000 shots of the MALDI laser

Supporting information Figure 3 Digestion of NLF by sialidase MALDI-TOF mass spectrometry of permethylated N-glycans from NLF after sialidase digestion (A, 1500-2334 m/z, B, 2333-3167 m/z and C, 3166-4250 m/z). Other conditions are the same as supplementary Figure E2. “M”, “m” and “vm” correspond to major, minor and very minor abundant structures. Data shown are a summation of spectra from 5000 shots of the MALDI laser.

Supporting information Figure 4 Digestion of NLF by endo-®-galactosidase MALDI-TOF mass spectrometry of permethylated N-glycans from NLF after endo-β-galactosidase digestion (A, 1500-2334 m/z, B, 2333-3167 m/z and C, 3166-4250 m/z). Other conditions are the same as supplementary Figure E2. Peaks that are not annotated correspond to enzyme resistant structures (same structures as displayed in Fig. 2A). Data shown are a summation of spectra from 5000 shots of the MALDI laser.

Supporting information Figure 5 Activation of dendritic cells by lactoferrin Day 8 BMDC at 2 x 106 cells/ml were incubated for 24 h with various concentrations of NLF or rice rLF (1-100 mg/ml) or with medium alone. Expression of the membrane markers MHC II, CD86 and CD80 were determined by flow cytometric analysis of 104 cells. Results are displayed as mean fluorescence intensity (MFI) recorded for each marker. Results from two independent experiments are shown.

Supporting Information Table 1 GC-MS linkage analyses of PMAA of NLF obtained after PNGAse F digestion.

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