Cathepsin X modulates profilin 1 function by affecting the binding of poly-L-proline ligands.

<p>(<b>A and B</b>) Profilin 1 forms a stable complex with actin and clathrin. Representative co-immunoprecipitations of profilin 1 with clathrin (A-inset) and actin (B-inset) in PC-3 cells, treated with DMSO or cathepsin X specific inhibitor. (<b>A and B insets</b>) Cell lysates were treated with anti-clathrin or anti β-actin antibody and immunoprecipitated on Protein A Sepharose beads. Profilin 1 was detected by Western blot in total cell lysates and in immunoprecipitated pellets. β-actin was used as a loading control. (<b>A and B</b>) Quantification of data from the insets. The graphs represent densitometric analysis of bands using Sygene's GeneTools Software (Sygene, U.K.). Three or two biological experiments indicate the normalized amount of profilin 1 that is in complex with clathrin (A) or actin (B), respectively. **P≤0.01. (<b>C</b>) Cathepsin X regulates actin polymerization. Flow cytometric analysis of permeabilized PC-3 cells is shown. Filamentous actin was stained with phalloidin conjugate. Cells were treated with DMSO or AMS-36 or were transfected with control or cathepsin X specific siRNA. Increase in actin polymerization is shown with values of both control experiments set to 0% increase in actin polymerization. Values are representative of four independent experiments. *P≤0.05; **P<0.01.