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Mannose Receptor-Mediated Regulation of Serum Glycoprotein Homeostasis
Sena J. Lee, Stefan Evers, Daniel Roeder, Albert F. Parlow, Juha Risteli, Leila Risteli, Y. C. Lee, Ten Feizi, Hanno Langen, and Michel C. Nussenzweig

Supplementary Material

Supplemental Figure 1. Generation of Mr-/- mice. (A) The maps of the genomic MR locus and targeting vector (EGFP, enhanced green fluorescent protein; Neo, neomycin-resistance selection marker; DTA, diptheria toxin A; P, Pst I). The EGFP reporter gene was inserted at the start codon in Exon 1, and it has a stop codon, which abolishes the expression of MR. (B) Embryonic stem cell screening for homologous recombination. A 300 bp probe upstream of Exon 1 was used for hybridization to Pst I-digested genomic DNA. The wild type (9.6 kb) and targeted (6.8 kb) fragments are indicated. (C) PCR genotyping of MR-/- mice. Two bands, 400 bp and 1.1 kb fragments, were amplified from the wild type and targeted allele, respectively. PCR amplification of the MR locus was done using the following PCR conditions and primers: 45 s at 94°C, 1 min at 55°C, 2 min at 72°C, 5�-GACCTTGGACTGAGCAAAGGGG-3� and 5�-GACATGATGTCCTCAGGAGGACG-3�. (D) Detection of MR expression by immunoblot. The anti-MR antiserum and HRP-conjugated donkey anti-rabbit IgG were used to detect MR protein from lymph node lysates. HRP-conjugated anti-Greek Letter Beta-actin antibody was used as loading control. For antibody production the cysteine-rich (CR) domain of the MR was expressed and purified as described (1). Rabbits were immunized with CR via intranodal injection (Covance Research Projects, Inc.) and the antisera were tested by immunoblot, using the purified CR and His-tagged CR proteins.

1. Y. Liu et al., J. Exp. Med.191, 1105 (2000).

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Supplemental Figure 2. Organ distribution of 125I-labeled Man-BSA. Fifty micrograms of 125I-labeled protein; specific activity of 2 � 10 cpm/Greek Letter Mug, was injected into the tail vein. Man-BSA (A and B), Gal-BSA (C), GlcNAc-BSA (D), GGnM-BSA and S4GGnM-BSA (E), Fuc-BSA (F), PICP (G), pLH (H). Mice were sacrificed at 10 (A, C, D, E, F, G) or 60 (B) minutes after injection. Each organ was excised and weighed, and the radioactivity of the samples was counted. The organ cpm values are expressed as the percentage of the total injected radioactivity found in each organ divided by the weight of the organ. Bars show the average values from three or more mice for each genotype, and error bars indicate +1 standard deviation (SD) values. (HR, heart; LG, lungs; LV, liver; SP, spleen; TH, thymus; GI, gastrointestinal tract).

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Supplemental Table 1.Greek Letter Beta-Glucuronidase levels were measured using fluorogenic enzyme substrate, 4-methylumbelliferyl-Greek Letter Beta-D-glucuronide, before and 2 days after intraperitoneal injection of 1ml 4% thioglycollate broth. Four mice per group were used. Greek Letter Beta-glucuronidase levels were calculated by dividing the mean fluorescence values from four mice per group by the blank controls (substrates only) and adjusting for total protein amount (ug) or serum volume (ul). SD(±1) values are also indicated.
After13.0±3.00 12.0±2.30