Effect of mAb L235 on cell migration. (A) Cell migration of HMEC-1, SK-MEL-28, or HUVEC cells was measured using modified Boyden chambers as described in “Materials and methods.” Cells that had migrated to the lower surface of the filters were fixed and stained with crystal violet. Images obtained from a representative experiment are shown. Cells that had migrated in the presence of 50 nM mAb L235 or a nonspecific mouse IgG were also counted. The results were expressed as the percentage of the control measured in the presence of a nonspecific mouse IgG and represent means ± SDs (n = 5 for HMEC-1; n = 4 for SK-MEL28; n = 3 for HUVEC). ***Statistically significant differences (P < .001; Student t test). (B) Detection of endogenous p97 by Western blot analysis. p97 was immunodetected in lysates or serum-deprived culture media (18 hours) from HMEC-1, SK-MEL28, and HUVEC-1 cells. Proteins were separated by SDS-PAGE and were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. p97 was detected by Western blotting, using mAb L235 and a secondary antimouse IgG linked to peroxidase.