Fig. 7.

Nck and ADAP are both required for actin rearrangement, as was detected in PBLs. (A) PBLs were treated with siRNA specific to Nckα,β or ADAP. After 48 h, cell lysates were prepared and analyzed for protein levels. The siRNA-mediated ADAP or Nck downregulation was compared to the level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase). (B) Nckα,β and ADAP knockdown experiments of were performed with human PBLs. PBLs pretreated with siRNA specific for Nckα,β and/or ADAP were seeded on stimulatory coverslips coated with anti-CD3 and anti-CD28. After 20 min of spreading, cells were fixed and stained with phalloidin (red). Overlay images and a summary of the results of 3 independent experiments (bottom) are presented. The actin spreading index was determined as described in Materials and Methods. t tests were calculated between cells treated with NS siRNA and the experimental groups. Gene silencing of both Nckα,β and ADAP caused a dramatic impairment of actin rearrangement in comparison to that in cells treated with NS siRNA (P < 0.000005). Slight impairments were detected in cells treated with Nckα,β or ADAP siRNA (P < 0.001 and P < 0.00001, respectively). t test analyses between the results for Nck or ADAP siRNA and a combined treatment with both Nck and ADAP siRNA were performed, and the P values were <0.00061 and 0.02, respectively.