Fig. 2.

Anti-CD148 monoclonal antibody (mAb; clone 18E1) inhibits cell proliferation of mouse CD148-expressing A431D cells, accompanied by an increase in CD148 catalytic activity. A: immunoblot analysis using 18E1 mAb. 18E1 mAb recognizes mouse CD148 (m-CD148, arrows), which is stably expressed in A431D cells (A431D/m-CD148) and wild-type (WT) kidneys, whereas its immunoreactivity is absent in control A431D cells (lacking CD148) and CD148 knockout (KO) mouse kidneys (left and middle). 18E1 mAb demonstrated CD148 expression in cultured mouse glomerular endothelial cells (Glom ECs), podocytes, and inner medullary collecting duct (IMCD) cells (right). The antibody was stripped, and the membranes were immunoblotted for actin (bottom) to assess the loading of protein lysates. Representative results of four independent experiments are shown. B: dose-dependent growth inhibition of A431D/m-CD148 cells by 18E1 mAb. A431D/m-CD148 or A431D cells were plated in 96-well plates and starved, and 10 μg/mL (left) or 1, 5, 10, or 20 μg/mL (right) of 18E1 mAb or class-matched IgG were then added to the medium. Cell number was assessed at days 0, 2, and 4. Data are means ± SE of quadruplicate determinations. Representative results of five independent experiments are shown. C: effects of CD148 knockdown in 18E1 mAb inhibition of A431D/m-CD148 cell proliferation. CD148 was knocked down in A431D/m-CD148 cells using lentivirus encoding mouse CD148-targeting shRNA (shRNA #1 and shRNA #2). Lentivirus encoding scrambled shRNA was used as a control. Their effects on 18E1 (10 μg/mL) inhibition of A431D/m-CD148 cell proliferation were assessed by a cell proliferation assay as in B. CD148 knockdown was confirmed by immunoblot analysis (top). Data show means ± SE of quadruplicate determinations. Representative data of four independent experiments are shown. **P < 0.01. Note that CD148 knockdown largely diminished 18E1’s growth inhibitory activity in A431D/m-CD148 cells. D: effects of 18E1 mAb on CD148 protein tyrosine phosphatase (PTP) activity. A431D/m-CD148 cells were treated with 10 μg/mL 18E1 or control IgG for 30 min with or without CD148-Fc (10 μg/mL) or Fc alone (1.2 μg/mL). CD148 was immunoprecipitated using anti-hemagglutinin (HA) antibody. As a control, immunoprecipitation was also conducted using control IgG. The washed immunocomplexes were subjected to a PTP activity assay with or without 1 mM sodium orthovanadate (VO₄). The amount of CD148 in the immunocomplexes was evaluated by immunoblot analysis using anti-HA antibody (top). Data show means ± SE of quadruplicate determinations. Representative data of five independent experiments are shown. **P < 0.01 vs. control IgG-treated cells. Note that CD148-Fc, but not control Fc, blocked 18E1 activity to increase CD148 catalytic activity.