After washing and blocking, the membranes were exposed in 1:2000-diluted serum for 1 h. The membranes were treated with 1:5000-diluted alkalinephosphatase-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). After incubation in a color development Selleckchem Berzosertib solution containing 0.3 mg/ml of nitroblue tetrazolium chloride (Wako Pure Chemicals) and 0.15 mg/ml of 5-bromo-4-chloro-3-indolylphosphate (Wako Pure Chemicals), positive reactions were detected. Positive clones were re-cloned twice to obtain
monoclonality. Sequence analysis of identified clones Monoclonalized phage cDNA clones were converted to pBluescript phagemids through in vivo excision using ExAssist helper phage (Stratagene, La Jolla, CA). Plasmid DNA was obtained from an E. coli SOLR strain transformed by the phagemid. The inserted cDNAs were sequenced using the dideoxy chain termination method and the sequences were analyzed for homology with a public database GS-4997 ic50 provided by the National Center for Biotechnology Information (NCBI). Production of glutathione S-transferase (GST) fusion proteins cDNA inserts of these clones incorporated in pBluescript were cleaved by EcoRI and XhoI generally and cloned into the EcoRI-XhoI site of pGEX-4 T-3, pGEX-4 T-2, and pGEX-4 T-1 see more vectors (Amersham Bioscience, Piscataway, NJ) that express recombinant
GST fusion proteins. E. coli JM109 cells containing pGEX clones (A600 = 0.3–0.5) were cultured in 200 ml of Luria broth (LB), and lysed through sonication. The lysate was then centrifuged and the
GST-fusion proteins in the supernatants were purified by glutathione-Sepharose. These samples were centrifuged and affinity-purified with glutathione-Sepharose. ELISA Purified recombinant proteins diluted at 10 μg protein/ml in PBS were added to each well of 96-well plates and incubated at room temperature overnight. As a control, the same amount of GST was applied. Sera diluted at 1:100 in PBS with 10% FBS were added to the wells and incubated for 1 h. The wells were exposed to 1:2 000-diluted horseradish peroxidase-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Cyclin-dependent kinase 3 West Grove, PA). Then, 100 μl of a peroxidase substrate (o-phenylenediamine, 0.4 mg/ml) containing 0.02% (v/v) H2O2 were added. Absorbance at 490 nm was determined using a microplate reader (Emax, Molecular Devices, Sunnyvale, CA). Construction of SH3GL1 deletion mutants Some deletion constructs of SH3GL1 were obtained through digestion with restriction enzymes or the inverse PCR method. The SEREX-identified phage clone was containing a full-length coding sequence of SH3GL1 (1–368 amino acids), that comprised Bin-Amphiphysin-Rvs (BAR) domain (amino acid positions between 5 and 242) in the N-terminal portion, coiled-coil (CC) domain (amino acid proteins between 180 and 250) at the middle, and the SH3 domain (amino acid positions between 309 and 364) in the C-terminal portion.