|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 4. p190 is required for multiple functional responses mediated by Sema4D in epithelial cells expressing endogenous PLXNB1. (A) Selective downregulation of p190 protein in SKBR3 cells by siRNA-mediated technology, demonstrated by immunoblotting with specific antibodies. The expression of endogenous PLXNB1 is unchanged. (B) Focal complex disassembly (revealed by vinculin immunostaining) and cellular collapse mediated by 1 hour treatment with 10 nM Sema4D was abrogated in p190-depleted SKBR3 cells. The functional response was unaffected by siRNAs targeting an unrelated sequence. The micrographs show results representative of at least three independent experiments. Scale bar: 40 µm. (C) Chemotactic migration of SKBR3 treated with siRNAs (as above) was assessed in Transwell® inserts, in the presence of 0.2 nM heregulin-ß1 (HRG), with or without 5 nM Sema4D. After 6 hours, the cells that had migrated across the porous membrane were stained with Crystal Violet. Cell migration was quantified by eluting the dye and measuring absorbance at 595 nm. Results shown are the average of two independent experiments, performed in duplicate. The expected inhibition of directional cell migration is lost in p190-deficient cells.
|
