Figure 3

Integrin clustering induced by CD98 cross-linking. RD A3 cells were grown on coverslips coated with fibronectin (20 μg/ml) for 48 hours. Cells were blocked with 20% goat serum and in some cases incubated with anti-CD98 mAb 4F2 (10 μg/ml) (C,D). Next, cells were washed, blocked with 20% goat serum and incubated with rabbit anti-mouse Ab (5 μg/ml). Finally, cells were fixed for 15 minutes with 2% paraformaldehyde, blocked with 20% goat serum and incubated with anti-α3 mAb A3X8 PUJ1 (10 μg/ml) (A, C) or anti-α4 mAb A4-PUJ1 (10 μg/ml) (B, D), each directly coupled with Alexa-488 (Molecular Probes). Coverslips were mounted with ProLong AntiFade Kit (Molecular Probes) and immunofluorescence examined using a Zeiss Axioscop equipped with optics for epifluorescence.