J. were recognized as substrates by all R3 members, but others were recognized by only one or a few members. The enzyme-substrate relationships identified in the present study will shed light on physiological roles of the R3 RPTP subfamily. and cDNAs were cloned by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA from mouse brain. Other RPTKs were cloned by RT-PCR using total RNA from human brain, spleen, and cell lines as templates. cDNAs for were cloned by RT-PCR using total Capromorelin RNA from mouse organs as templates. Substrate-trapping DA RPTP mutants, in which a conserved aspartic acid in the active center was substituted with alanine, were constructed by site-directed mutagenesis. The intracellular regions (ICRs) of were subcloned into the vector pCMV-BD (Stratagene) to produce fusion proteins comprising the Gal4 DNA-binding domain and a RPTK ICR (BD-RPTKs). The ICRs of were subcloned into the vector pCMV-AD (Stratagene) to produce fusion proteins comprising the NF-B Capromorelin activation domain and a RPTP ICR (AD-RPTPs). The ICRs of were also subcloned into the vector pGEX-4T (GE Healthcare) to produce fusion proteins with glutathione were subcloned into the expression vector p3xFLAG-myc-CMV-23 (Sigma) to produce FLAG-tagged RPTK proteins (FLAG-RPTKs). The full-length cDNA of mouse was subcloned into the expression vector pcDNA-Myc to produce Myc-tagged EphB2 protein (EphB2-Myc). The full-length were subcloned into the expression vector pDisplay (Invitrogen) to produce HA-tagged RPTP proteins (HA-RPTPs). The DNA fragments encoding HA-tagged full-length from pDisplay-RPTP plasmids were also subcloned into the expression vector pBABE-puro, which contains a puromycin resistance gene, for the cell proliferation assay. Short Hairpin RNA (shRNA) Constructs The pBAsi-hU6 Pur vector (Takara Bio, Shiga, Japan), which contains a puromycin resistance gene, was used to express shRNAs to knockdown the expression of targeted genes in HUVEC-C cells. To construct shRNA vectors, the following oligonucleotide DNAs were inserted into BamHICXbaI sites of the pBAsi vector: Control scrambled shRNA, 5-GATCCGCTGCTCTAGGTTGTCAGGTATGTGTCATGACGATTGGTACTAAGGACTAGGAGCTTTTTTA-3 and 5-CTAGTAAAAAAGCTCCTAGTCCTTAGTACCAATCGTCATGACACATACCTGACAACCTAGAGCAGCG-3; dephosphorylation, we first prepared autophosphorylated RPTK proteins as substrates. A FLAG-RPTK (or Capromorelin Eph-Myc) was transfected into HEK293T cells. After 24 h, cells grown on a 35-mm culture dish were lysed with RIPA buffer, and the lysates were subjected to immunoprecipitation with various antibodies bound to Protein G-Sepharose (BD Healthcare). Protein G beads were then washed once and resuspended in 100 l of 10 mm Tris-HCl, pH 7.0, containing 5 mm DTT, 5 mm EDTA, and 100 g/ml of bovine serum albumin (PTP buffer). For the dephosphorylation assay, 10 ng of GST-RPTPs or GST alone was reacted with 10 l of RPTK solutions at 30 C for 30 min. The samples were separated by SDS-PAGE, followed by immunoblotting with specific primary antibodies and peroxidase-linked secondary antibodies, and visualized by chemiluminescence using ECL Reagent. Coexpression Assay of RPTK and RPTP A (or into HEK293T cells grown on a 35-mm culture dish. After 24 h, cells were lysed in RIPA Pax6 buffer, which consists of 20 mm Hepes, pH 7.0, 120 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 0.05% SDS, 50 m Na3VO4, and a protease inhibitor mixture (1 g/ml of pepstatin A, 10 g/ml of leupeptin, and 1 mm phenylmethylsulfonyl fluoride). Cell lysates were clarified by centrifugation, and subjected to immunoprecipitation with various antibodies bound to Protein G-Sepharose (BD Healthcare). Immunoprecipitates were treated with SDS sample buffer and subjected to SDS-PAGE. Proteins were transferred onto Immobilon-P membranes (Millipore), reacted with specific primary antibodies and peroxidase-linked secondary antibodies (BD Healthcare), and visualized by chemiluminescence using ECL Reagent (BD Healthcare). The lumino-image analyzer LAS-1000 (Fujifilm) was used for the detection. Signal intensity was quantified and analyzed by Student’s test. Down-regulation of RPTPs in HUVEC-C Cells The pBAsi shRNA expression plasmids were introduced into cells using Lipofectamine Plus according to the manufacturer’s protocol. To select plasmid-containing cells, 12 h after transfection, puromycin was added to the culture medium at 2 g/ml. After 24 h incubation, cells were re-plated at 5 104 cells per 35-mm dish in the medium containing 0.5% FBS. After another 12-h incubation, cells were treated with basic FGF (20 ng/ml, Wako), or ephrin-A2-Fc proteins (10 g/ml) (6) for 10 min. Then, cells were analyzed by Western blotting similar to the RPTK/RPTP coexpression assays..