Fig. 1.

EGFR of A431 cells expresses glycan(s) with terminal GlcNAc structure, probed by mAb J1 and by lectin GS-II. A431 cells were grown, harvested, pelleted, and lysed in RIPA buffer, and lysate was subjected to Western blot analysis as described in Materials and Methods. (A) EGFR band (≈170-kDa molecular mass) blotted by anti-EGFR mAb, mAb J1, and HRP-labeled GS-II lectin. Protein of cell lysate [25 μg (lane 1), 50 μg (lane 2), or 100 μg (lane 3)] was analyzed by Western blot with the respective Abs and lectin as indicated on the right. (B) GS-II/agarose binding moiety of lysate, Western blotted by anti-EGFR mAb and mAb J1. We washed 25 μl (lane 1), 50 μl (lane 2), and 100 μl (lane 3) of GS-II/agarose beads (4–5 mg of GS-II per milliliter of beads) twice with ice-cold PBS and incubated them with A431 cell lysate (0.5 mg of protein) in PBS containing 0.9 mM CaCl₂, 0.5 mM MgSO₄, and 0.1 mM MnCl₂ with tumbling for 1 h at 4°C. After centrifugation at 1,410 × g for 5 min at 4°C, the beads were washed three times with 1 ml of PBS containing 0.9 mM CaCl₂, 0.5 mM MgSO₄, and 0.1 mM MnCl₂, followed by centrifugation at 1,410 × g for 5 min. The bound fraction was released by boiling in SDS/PAGE sample buffer and analyzed by Western blot with anti-EGFR mAb and with mAb J1. (C) Absence of mouse IgG in A431 cell lysate. Because EGFR was probed by anti-human EGFR mouse IgG mAb, the possible presence of mouse IgG in original A431 cell lysate should be ruled out. For this purpose, A431 cell lysate (500 μg of protein) was precleared with 25 μl of protein A/G-agarose beads for 2 h at 4°C and centrifuged at 980 × g for 5 min at 4°C. The supernatant was incubated with 4 μg of anti-EGFR (lane 1) or 4 μg of mouse IgG (lane 2) as a control overnight at 4°C. The supernatant was then added to 25 μl of protein A/G-agarose, tumbled for 2 h at 4°C, and centrifuged at 980 × g for 5 min at 4°C. The beads were washed twice with 1 ml of RIPA buffer, and the samples were prepared as above for SDS/PAGE and Western blot with anti-EGFR mAb and mAb J1. Note that immunoprecipitate by anti-EGFR (lane 1) but not by mouse IgG (lane 2) showed reactivity with anti-EGFR and J1. Arrows on the left indicate ≈170 kDa based on the positions of molecular mass markers.