Bivalent Ab1 suppresses phosphorylation of ERK1/2 and Met kinases with an increase in CD148-associated PTP activity. (A) HRMECs and the CHO cell line (CHO-CD148), which is stably expressing CD148, were serum starved for 24 hours and 72 hours, respectively; pretreated with 67 nM Ab1 (bivalent) or Ab1.Fab (monovalent); and stimulated with medium supplemented with 2.5% FBS in the presence of Ab1 or Ab1.Fab (67 nM). The cell lysates were subjected to immunoblots using phospho-peptide–specific ERK1/2 (p-ERK) and Akt (p-Akt) antibodies. Blots were stripped and reprobed for total ERK1/2 (ERK) and Akt (Akt). (B) Serum-starved subconfluent HRMECs were pretreated with Ab1 or Ab1.Fab (67 nM), and then stimulated with 10 ng/mL HGF in the presence of Ab1 or Ab1.Fab (67 nM). Met kinase (HGF receptor) was immunoprecipitated from the cleared lysates and its phosphorylation level was examined by immunoblots using a phospho-peptide–specific Met antibody (p-Met) as described in “Materials and methods.” Blots were stripped and reprobed for total Met (Met). (C) HRMECs were plated on 100-mm dishes, serum starved, and incubated with either Ab1 or Ab1.Fab or control IgG₁ (67 nM) for 15 minutes. The cells were lysed in buffer and CD148 was immunoprecipitated using affinity-purified CD148 rabbit antibody or control rabbit IgG. The washed immunocomplexes were assayed for PTP activity with or without 1 mM sodium orthovanadate (VO4) as described in “Materials and methods.” The data are presented as means ± SEM of quadruplicate determinations.