0.4. -AGEs IgM titer AGEs IgM 0.4

Transcription

1 IgG titer (Absorbance at 49nm) C IgG titer (Absorbance at 49nm) IgG IgG (Absorbance at 49nm) D (Absorbance at 49nm) IgM IgM Fig. S1. Presence of natural antibodies against the DHA- in normal male C57L/6 mice (6-week-old) and male mice (18-week-old). (a) The titer of IgG antibodies in the C57L/6 mice sera to the antigens. (b) The titer of IgM antibodies in the C57L/6 mice sera to the antigens. (c) The titer of IgG antibodies in the mice sera to the antigens. (d) The titer of IgM antibodies in the mice sera to the antigens. The IgG and IgM titers in the mouse sera were examined by an ELISA employing pairs of wells in microtiter plates on which were absorbed native, DHA-modified (DHA-), and as the antigens.

2 (Absorbance at 49nm) ds 5 1 Fig. S2. Presence of natural antibodies against the DHA- in SPF-maintained alb/c mice (8-week-old). The titer of IgM antibodies in the SPF mice sera to the endogenous antigens. The s in the mouse sera were examined by an ELISA employing pairs of wells in microtiter plates on which were absorbed native, DHAmodified (DHA-), and as the antigens.

3 nti-dha- IgM (Absorbance at 49nm) Incubation (days) Fig. S3. Cross-reactivity of the sera from and mice with DHA-treated proteins. (1. mg/ml) was incubated with DHA (25. mm) in PS buffer (ph 7.4) at 37 C under atmospheric oxygen. After 1, 3, 5, 7, and 14 days, aliquots were collected and the IgM response was examined by an ELISA employing pairs of wells in microtiter plates on which were absorbed DHA-treated (DHA-) as the antigen.

4 IgG titer (Absorbance at 49nm) DHA- (Absorbance at 49nm) DHA- Fig. S4. Immunoreactivity of IgG and IgM antibodies eluted from the kidneys of and mice. (A) IgG titer. (). The kidneys were thawed, minced, and homogenized in a high-speed blender for 1 min in 4 volumes of cold PS, and then repeatedly washed in PS. The washed homogenates were then incubated with 1 ml glycine-hci buffer (2 mm, ph 3.) at room temperature for 1 min, centrifuged at 1,5 rpm, and the ph of the supernatant adjusted to ph 7.4. The eluates were dialysed overnight against PS at 4 C. The antibody titer was determined by a direct antigen ELISA using native and DHA-modified (DHA-), and as the absorbed antigens.

5 DHA (mm) Trimer Dimer Monomer + Fig. S5. PAGE analysis of DHA-modified proteins. (1 mg/ml) was incubated with DHA (-25 mm) in 5 ml of PS, ph 7.4, for 7 days at 37 C.

6 (absorbance at 49nm) ssdna ssrna dsrna poly(i:c) (absorbance at 49nm) GpC ODN CpG ODN C (absorbance at 49nm) PE PC PS IPP Fig. S6. Cross-reactivity of the sera from and mice with various electronegative molecules. Serum samples from 27-week-old and mice were used. (A) Cross-reactivity with nucleic acids. The nucleic acids used were single-stranded DNA (ssdna), (double-stranded DNA), ssrna (polyuridylic acid potassium salt), dsrna, and poly(i:c) (a synthetic analog of dsrna). () Cross-reactivity with unmethylated CpG oligodeoxylibonucleotide (ODN) and its negative control GpC ODN. (C) Cross-reactivity with phospholipids and a phosphoantigen, isopentenyl pyrophosphate (IPP). The phospholipids used were phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylserine (PS). In panels A-C, the s were determined by ELISA. Poly (I:C) was obtained from InvivoGen. CpG ODN and GpC ODN were obtained Imgenex. Other nucleic acid and phospholipid antigens were obtained from Sigma.

7 3. 2. C 2. (absorbance at 49nm) (absorbance at 49nm).8.4 (absorbance at 49nm).8.4 ssdna ssrna dsrna poly(i:c) GpC ODN CpG ODN PE PC PS IPP Fig. S7. Cross-reactivity of the anti- IgM DM1 with various electronegative molecules. (A) Cross-reactivity with nucleic acids. () Cross-reactivity with unmethylated CpG ODN and its negative control GpC ODN. (C) Crossreactivity with phospholipids and IPP. In panels A-C, the s were determined by ELISA. The sources and abbreviations of antigens used in the experiments were described in the legend of Fig. S6.