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Blood, Vol. 108, Issue 4, 1234-1242, August 15, 2006
A monoclonal antibody against CD148, a receptor-like tyrosine phosphatase, inhibits endothelial-cell growth and angiogenesis
Blood Takahashi et al.
108: 1234
Supplemental materials for: Takahashi et al
Files in this Data Supplement:
- Figure S1. siRNA-mediated knock-down of CD148 increases cell proliferation in CHO-CD148 cells (JPG, 15.9 KB)
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(A) The CHO cell line stably transfected with HA-tagged CD148 (CHO-CD148) was transiently transfected with 100 nM of CD148 short interfering RNA (siRNA) using Gene Eraser siRNA transfection reagent (Stratagene, La Jolla, CA). Two days after transfection, total cell lysates were subjected to immunoblotting with anti-HA and anti-γ-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA). The CD148 siRNA duplexes (5′-GCAGUACAGCAGAAUCCUU-3′ and 5′-TCGUCAUGUCGUCUUAGGAA-3′) were synthesized by Dharmacon (Lafayette, CO) and a scrambled siRNA duplex (Dharmacon, Lafayette, CO) was used as a control.
(B) CHO-CD148 cells were transfected with the indicated siRNA and replated on 12 well plates at 24 hours after transfection (day 0), and cultured in growth media supplemented with 2.5% FBS. The cell number was evaluated at indicated time points by a crystal violet assay. Plots indicate mean values ± SEM. of quadruplicate determinations.
- Figure S2. EGFR-CD148 chimera inhibits 32D cell proliferation on EGF stimulation (JPG, 46.9 KB)
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(A) Expression plasmids of epidermal growth factor receptor (EGFR)-CD148 chimera were generated by linking ecto- and transmembrane domains of human EGFR to CD148 cytoplasmic domain (wild-type, c-s mutant). The plasmids were introduced to 32D cells which lack EGFR by electroporation (BIORAD, Gene PulserII), and stable cell lines were prepared by neomycin (750µg/ml) selection50. The cell lines which express comparable surface level of EGFR-CD148 chimera were selected by FACS using anti-EGFR antibody (kindly provided from Dr. J Staros)50 and used for the study in panel B. (B) Cells were starved for 18 hours in the absence of FBS and IL-3, and 1 × 105 cells were seeded in T25 flask with RPMI 1640 medium supplemented with 2.5% FBS, 5% IL-3, and EGF (0, 5, 25 µg/ml) (day 0). The medium was replaced every 2 days. Cells were counted at days 1, 2, and 4.
- Figure S3. High cell density increases CD148 dimerization and catalytic activity (JPG, 88.5 KB)
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(A) HRMEC (5 × 105) cells were plated on100 and 35 mm dishes to generate the differences in cell density. After 24 hours, the cells were washed with PBS and incubated with freshly prepared 1mM DTSSP cross-linker (Pierce, Rockford, IL) for 1 hour on ice. After washing with PBS, the cells were incubated in 20mM Tris /pH7.5 for 15mins on ice to quench the reaction, and lysed in buffer (50mM HEPES, 1% Triton, 150mM NaCl, 1.5mM MgCl2, 1mM EGTA, 10% glycerol, protease inhibitor cocktail). The clarified protein lysates (300 µg) were subjected to immunoprecipitation with affinity-purified CD148 rabbit antibody6. The immunocomplexes were separated by SDS-PAGE on 5% polyacrylamide gels under non-reducing (left) or reducing (right) conditions and immunoblotted with Ab1 antibody. A CD148/Fc fusion protein (440kDa) was used as a size marker. CD148 monomer is indicated by asterisks and a higher order CD148 oligomer (potential dimer) by an arrow respectively.
(B) HRMEC cells were plated on100 and 35 mm dishes, lysed in buffer (50mM HEPES/pH7.5, 150mM NaCl, 5mM EDTA,1% TritonX-100, 1mM DTT, 5µg/ml aprotinin, 1 µg/ml leupeptin), and CD148 was immunoprecipitated from the protein lysates (150µg) using affinity-purified CD148 rabbit antibody. Washed immunocomplexes were assayed for PTP activity as described in Methods. Phosphatase activity was measured with or without 1mM sodium orthovanadate (VO4). The data is presented as mean ± SEM of quadruplicate determinations.
(C) FRET interaction of CD148-YFP and CD148-CFP in CHO cells. The EYFP and ECFP sequences were c-terminally fused to human CD148. The CD148 (wt)-YFP and CD148 (wt)-CFP plasmids were transiently co-transfected into CHO cells which lacks endogenous CD148 expression. FRET signals in living cells were evaluated by confocal microscopy. Following excitation of the donor fluorophore (CFP) with the 428nm, YFP fluorescence emission was observed by confocal microscopy (panel A). Note: YFP fluorescence is emitted at intercellular sites (arrow) by CFP emission, indicating FRET interaction (cis-interaction of CD148-YFP and CD148-CFP), in CD148-YFP&CD148-CFP transfected cells but not in CD148-YFP cells. FRET signals were also evaluated by bleaching YFP (lower panels). Circles indicate the bleached areas. Note: Bleaching YFP fluorescence increases CFP fluorescence in CD148-YFP&CD148-CFP transfected cells (arrow, 250% increase) but not in CD148-CFP transfected cells.
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