FIGURE 3.

Metalloproteinase-mediated ectodomain cleavage of the receptor isoforms of Ptprz. A, HEK293T cells were transiently transfected with the expression construct of Ptprz-B or control vector (Moc). Twenty-four hours after transfection, cells were washed and incubated with or without PMA in fresh serum-free medium for 1 h. GM6001 was added 20 min before the stimulation by PMA or vehicle. The cell extracts (left panels) were analyzed by Western blotting (WB) using anti-Ptprz-S. The same membrane was then reprobed with anti-RPTPβ. Conditioned media (right panel) were analyzed with anti-Ptprz-S. B, cells were incubated with the indicated amount of PMA in fresh serum-free medium for 1 h and analyzed by Western blotting as above. C, HEK293T cells were transiently transfected with the Ptprz-A or Ptprz-S expression construct and treated as described in A. Before SDS-PAGE, the samples were treated with chABC. The figures are representative of three separate experiments, and the results of the densitometric analyses are shown in supplemental Fig. S1. The designations of the detected bands are shown in Fig. 1, A and E.