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Fig. 7. EGFR regulates the migration of uPAR-wt keratinocytes by controlling LN5 deposition. (A) Migration of keratinocytes was analyzed by 48-well Boyden chamber assay in S-MEM 1% chelexed serum with or without AG1478 (10 µM) through FN-, ColIV-, VN- or LN5-coated 8-µm-pore-size polycarbonate filters at 37°C for 6 hours. The mean number of migrated cells ± s.d. from triplicate samples is representative of at least three experiments. (B,C) Suspended wt and uPAR-KO keratinocytes were seeded on a ColIV-coated surface with or without AG1478 (10 µM). After 45 minutes, attached cells were detached with 10 mM EDTA, used for mRNA isolation, and subjected to RT-PCR using LN5 (
2 chain) primers (B), whereas secreted LN5 was detached from the surface with SDS-sample buffer and immunoblotted (IB) with anti-LN5 (2 chain) antibodies (C). Actin was used as a control. The amounts of secreted LN5 (2 chain) (protein and mRNA) were quantitated and expressed relative to levels in wt samples (=100%) ± s.d., and are representative of at least three experiments.
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