Figure 3

No dissociation of feline AIM from IgM pentamer.

(a) Immunohistostaining of the kidney at day 1 after IR. Kidney specimens of AIM⁻ mouse were also immunostained for mouse or feline AIM. Signals were visualised by HRP/DAB. Five mice each were analysed. (b) Immunoblotting in non-reducing conditions for feline AIM (AIM felinised mice; n = 3), and mouse AIM (wild-type mice; n = 2). Pre: before IR. 3-SRCR feline rAIM (15 ng) and mouse rAIM (20 ng) were loaded as controls. (c) Immunoblotting of 4 IR cats in a non-reducing (upper panel) or reducing (lower panel) condition for AIM. Genotype: #1 and 2, 3/4-SRCR; #3, 3/3-SRCR, #4, 4/4-SRCR. rAIM (15 ng each) were loaded as controls. A longer-exposed blot is in Supplementary (b). (d) Immunostaining for feline AIM at the corticomedullary junction in the kidney of an IR cat (#3) at day 2. Scale bars, 50 μm. Black arrows: intraluminal debris. Results from other IR cats are in Supplementary (d). (e) Relative KIM-1 mRNA levels in the kidney of 3 cats after IR. Kidneys from healthy cats (26 months of age, male, n = 2) were analysed as controls. (f) SPR analysis for AIM (as analyte) and IgM pentamers (as ligand). Three sets of experiments were performed and similar results were obtained. (g) Immunoblotting in a non-reducing (upper panel) or reducing (lower panel) condition for feline AIM. Two different sizes of ΔSRCR-3 are indicated by arrows. Wild-type: wild-type feline AIM (3-SRCR). (h) Three-dimensional mapping of the charge distribution of amino acids for the SRCR3 domain. Positively charged: blue; negatively charged: red; neutrally charged: white. The feline AIM-specific cluster of positively charged amino acids is gated. Feline-specific arginine residues, and the corresponding amino acids in mouse, human and mutant feline AIM, are demonstrated. (i) Binding of wild-type, chimeric, and mutant feline AIM with feline IgM Fc pentamer assessed by immunoblotting (non-reducing conditions). Signal intensities of the IgM-Fc bound AIM were quantified using NIH ImageJ software, and the relative values to that in wild-type feline AIM are presented in a graph.