Mouse serum ferritin is not detectably glycosylated. (A) Deglycosylation of immunoprecipitated liver and serum ferritin from iron-overloaded mice with Peptide: N-Glycosidase F (PNGase-F) followed by separation on SDS-PAGE and Western blot with an anti–mouse liver ferritin antibody. Lanes 1 and 2 are pre-clears before and after deglycosylation, respectively. Lanes 3 and 4 are immunoprecipitated samples before and after deglycosylation, respectively. No change in molecular weight was detected in L- ferritin subunit after PNGase F digestion. Note the significant amount of the S-subunit detected in serum, compared with liver ferritin. An internal positive control is marked with arrowheads and shows that IgG heavy chain was successfully deglycosylated (1 representative experiment is shown of 5). (B) ConA binding of mouse serum ferritin. Serum was incubated with protein A sepharose and with ConA beads (lanes 1 and 3, respectively) and their supernatants were immunoprecipitated with anti–liver ferritin antibody (lanes 2 and 4). Serum was directly immunoprecipitated with anti–liver ferritin antibody (lane 5) and ConA beads were loaded in lane 6. All samples were analyzed on SDS-PAGE followed by Western blot analysis (1 representative experiment is shown of 5).