|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 4. uPAR stimulation results in Src-dependent phosphorylation of caveolin at Tyr14. (A) Confluent fibroblast monolayers were treated with either 50 µM of the uPAR ligand, P25, or the control peptide, S25, for 1 hour. Cell layers were lysed in sample buffer. Lysates were electrophoresed, immunoblotted and analyzed using an antibody that recognizes caveolin phosphorylated at Tyr14 (caveolin PY14). Blots were then stripped and re-probed with a total caveolin antibody to verify equal loading. (B) Cells were transfected with uPAR siRNA or a non-targeting siRNA for 72 hours. Cell layers were lysed in unreduced sample buffer. Lysates were electrophoresed and immunoblotted using an antibody against uPAR or Erk2. (C) Gels were scanned and uPAR values were normalized to Erk2. Values from control siRNA cells were set at 1. Bars reflect the range of knockdown (n=2). (D) Cells treated with either non-targeting siRNA or uPAR siRNA were incubated with 50 µM P25 or S25 for 1 hour. Cell layers were lysed in sample buffer and lysates were analyzed by western blot for caveolin PY14. Blots were stripped and re-probed for total caveolin to verify equal loading. (E) Fibroblast monolayers were pre-treated with 10 µM PP2 or 5 µM AG1478 (AG) for 1 hour before treatment with 50 µM P25 or S25. Cell lysates were electrophoresed and immunoblotted using an antibody against caveolin PY14. Blots were then stripped and re-probed for total caveolin. (F) Cells were treated with P25 or S25 as described in A and then extracted in RIPA buffer. uPAR was immunoprecipitated from lysates using anti-uPAR antibody 3937. Immune complexes were precipitated with protein A/G agarose beads, solubilized in unreduced sample buffer and electrophoresed into gels. Components of the complexes were analyzed by western blotting.
|
