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. 2006 Dec 1;103(50):18987–18991. doi: 10.1073/pnas.0609281103

Fig. 2.

Fig. 2.

Binding of GM3 and other GSLs to EGFR and inhibition of this binding by Os Fr.B. (A) Isolation and characterization of structure (see ref. 21). Structure of Os Fr.I and Os Fr.B. Os Fr.B is a mixture of structures with and without Galβ4 substitution to a defined N-acetylglucosamine (Gn) as shown. M, mannose. (B) Interaction of GSL-coated polystyrene beads with EGFR and inhibition of this interaction by Os Fr.B. GM3, GM1, or Gb4 (16 nmol each) was dried, dissolved in 1 ml of an ethanol/water solution (9:1), mixed with 6.85 × 107 polystyrene beads [1 μm in diameter (Interfacial Dynamics, Portland, OR), previously washed in PBS, and suspended in ethanol], and incubated with tumbling for 1 h at room temperature. After being washed three times with 0.5 ml of PBS, the beads were incubated with 300 μl of 0.1% gelatin for 1 h with tumbling at room temperature to block nonspecific binding sites, centrifuged at 1,410 × g for 5 min, washed with TBS, and resuspended in 500 μl of TBS(+) [130 mM NaCl/10 mM Tris·HCl (pH 8.0) containing 0.9 mM CaCl2, 0.5 mM MgSO4, and 0.1 mM MnCl2] containing A431 cell lysate (30 μg of protein). The mixture was kept overnight at 4°C with tumbling and then washed three times with TBS(+). Protein bound to GSL-coated beads was solubilized by boiling in SDS/PAGE sample buffer as above and analyzed by Western blot with anti-EGFR mAb. For effects of OS Fr.B, OS Fr.I, or cellobiose on binding of EGFR to GM3-coated beads, beads were resuspended in 500 μl of TBS(+) containing A431 cell lysate and 16 nmol of Os Fr.B (molecular weight, 2,211.3), 16 nmol of Os Fr.I (molecular weight, 1,317.4), or 16 nmol of cellobiose (molecular weight, 342.3). For a control, GSL-noncoated beads were incubated with A431 cell lysate in the absence of Os or cellobiose. EGFR bands in the Western blots in each lane are as follows. a, beads alone; b, beads coated with GM1; c, beads coated with Gb4; d, beads coated with GM3; e, beads coated with GM3 and incubated with cellobiose; f, beads coated with GM3 and incubated with Os Fr.B. Beads coated with GM3 and incubated with Os Fr.I showed reduced adsorption of EGFR, as in our previous study (ref. 21 and data not shown).