Fig 1.
Comparison of AF20 antigen levels in different cell lines.
Cell lysate containing 200μg of proteins was subject to immunoprecipitation with AF20 antibody followed by Western blot using the same antibody. The 50-kDd protein band in the blot corresponds to the heavy chain of AF20 antibody (IgG). Pro RG: proliferating HepaRG cells. Note that for the left panel, lane 1 and lanes 2/3 were cropped from different parts of the same blot.
Fig 2.
Purification of AF20 antigen from Huh7 cells for proteomic analysis.
A. Cell pellet (2 grams) was lyzed by freeze/thaw and diluted with Tris buffer. The sample was loaded onto a DEAE-cellulose column. After flow through (FT), the column was washed with Tris buffer (wash). Proteins were eluted successively with 100 mM, 200 mM and 400 mM of NaCl, and protein concentration was determined by BCA assay using known concentrations of albumin as a standard. (B & C) AF20 antigen was imunoprecipitated from the three protein peaks by AF20 mAb and subjected to Western blot (minigel format) using the same antibody (B), or pooled and subject to SDS-PAGE (large gel format) followed by Coomassie blue staining (C). The area of three protein bands was cut out for proteomic analysis. MW: molecular size markers.
Table 1.
Protein sequencing results.
Fig 3.
AF20 antibody recognizes TFR1 but could co-immunoprecipitate HSP90 and Na+/K+ ATPase from Huh7 cells.
A. Huh7 cells were transiently transfected with DDK tagged TFR1 cDNA. Lysate of transfected or non-transfected cells was immunoprecipitated with DDK or AF20 mAb, followed by sequential detection of the blot with DDK and TFR1 antibodies. B. Lysate of nontransfected Huh7 cells was incubated with AF20 mAb immobilized on protein G beads, or protein G beads alone to serve as a negative control, followed by Western blot detection of beads-associated proteins by antibodies against AF20, TFR1, HSP and N+/K+ ATPase, respectively. Note that a minigel was used for electrophoresis, which could not resolve proteins of similar sizes.
Fig 4.
Affinity of AF20 mAb for TFR1 but not Na+/K+ ATPase or HSP90 by immunofluorescent staining.
NIH 3T3 cells grown in 24-well plate with cover slips were transfected with expression constructs of DDK tagged TFR1, DDK tagged Na+/K+ ATPase, or untagged HSP90. Cells were fixed with acid alcohol followed by staining with AF20, TFR1, DDK, and HSP90 antibodies, respectively. Note that AF20 mAb only revealed signals in TFR1 transfected cells, although all three proteins were successfully expressed in NIH 3T3 cells. In addition, cell death or unusual morphology (elongated) was observed in cells expressing the exogenous proteins (dead cells are indicated by arrows). All images were taken at 20x magnification.
Fig 5.
Subcellular localization of TFR1 as revealed by AF20 or TFR1 mAb.
TFR1 transfected NIH 3T3 cells were fixed and stained with either AF20 mAb (panel A) or TFR1 mAb (panel B), followed by confocal microscopy to reveal subcellular localization and 3D pattern of the protein. (C) Stacks of X axis for panels A and B, respectively.
Fig 6.
Holo but not apo transferrin could interfere with AF20 antigen-antibody interaction.
A. Lysate of LS180 cells was subjected to immunoprecipitation with AF20 mAb in the absence (w/o) or presence of 33μg/ml of apo or holo transferrin (TF), and retained proteins were detected by Western blot with AF20 mAb. B. Increasing concentrations (3.3, 16.5, and 66μg/ml) of holo transferrin were added during immunoprecipitation with AF20 mAb, followed by Western blot with the same antibody.
Fig 7.
AF20 mAb failed to recognize deglycosylated TFR1.
Huh7 cell lysate was subject to immunoprecipitation with AF20 mAb and retained proteins on protein G beads were treated with PNGase F at 37°C for 1hr. Samples were directly loaded onto 10% SDS-PAGE (minigel) in duplicate followed by Western blot detection using AF20 (left) and TFR1 antibodies (right), respectively. Note that an additional protein band above the 75-kd size marker was detected by TFR1 antibody from PNGase F treated sample, most likely corresponding to deglycosylated form of TFR1 (79kDd).
Fig 8.
Correlation of AF20 expression with malignant transformation of colon tissues.
A total of 4 pairs of tissue sections were stained with AF20 antibody followed by incubation with HRP-conjugated anti-mouse antibody. Two representative pairs are shown (upper and lower panels). Note that AF20 was clearly detectable in colon polyps (middle panels), and strongly expressed in colon cancer (right panels), but not in normal colon tissue (left panels). Images were taken at 20 x magnifications, with the scale bar provided.
Fig 9.
Correlation of TFR1 and AF20 staining in colon cancer samples.
Three pairs of adjacent tissue sections of normal colon (A) and colon cancer (B) were stained with TFR1 (upper panels, 1:50 dilution) and AF20 mAb (lower panels, 1:500 dilution), respectively. Images were taken at 1000x magnification. Note that overexpression of both TFR1 and AF20 antigen was only observed in colon cancer but not in normal colon. In addition, the expression pattern and localization as revealed by TFR1 and AF20 Ab are indistinguishable.