Analysis of Sera of Recipients with Allograft Rejection Indicates That Keratin 1 Is the Target of Anti-Endothelial Antibodies

Figure 3

Antibody elution and EC target antigen immunoprecipitation. (a) Sera S1, S3, and S5 with anti-HLA, anti-MICA, and anti-EC antibodies, respectively, as well as normal human serum were incubated with HUVECs. HUVECs were lysed, and the antibody-antigen complex was captured using protein-G magnet beads. Precipitated proteins were separated on SDS-PAGE, and immunoblots (IB) were performed using HC10 (anti-HLA heavy chain), 1.7AD (anti-MICA), and anti-human IgG, respectively. (b) HUVECs were incubated with serum S5 (top), supernatant after elution step (middle), and serum antibodies were enriched by antibody absorption and elution as shown in Figure 2, left. (c) Proteins precipitated with S5 were separated on SDS-PAGE and revealed by silver staining. Normal human serum was used as negative control. The captured antigen from HUVECs and IgG bands are indicated.

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