316–368, 260–289 and 354–368 were deleted through PI3K inhibitor the inverse PCR method with the KOD–Plus Mutagenesis Kit (Toyobo, Osaka, Japan) using the SH3GL1 mut-1 cDNA as a template (SH3GL1 mut-2, 3 and 4, respectively). The primers for SH3GL1 mut-2 were forward 5′-CCAGTCTTCCGACAAGCCCATC-3′, reverse 5′-TGGGGATCCACGCGGAACCAG-3′; for SH3GL1 mut-3 were forward 5′-TCGAGCGGCCGCATCGTGAC-3′, reverse 5′-GCCCGACTGGCCGTCCAGCATG-3′; and for SH3GL1 mut-4 were; forward 5′-TCGAGCGGCCGCATCGTGAC-3′, reverse 5′-GCCCGACTGGCCGTCCAGCATG-3′. Overlap peptide array Peptides spanning amino acid residues 1–368 of SH3GL1 were synthesized on cellulose membranes as a series of peptides with the overlapping by 12 amino acids using F-moc amino acids according to the manufacturer’s protocol (Auto spot robot ASP222; ABIMED Analysen-Technik GmbH, Langenfeld,
Germany) as previously described . Membranes were incubated with the sera of patients at 1:200 dilutions for more than 12 h. Then, the antigen-antibody complexes were detected with FITC-conjugated STA-9090 goat anti-human IgG (109-095-098; Jackson ImmunoResearch, West Grove, PA) at 1:10000 dilutions. The fluorescence of the peptide spots were detected using Typhoon 9400 (Amersham Biosciences, Stockholm, Sweden) with a 488 nm/520 nm filter. The scanned image was also analyzed with CS analyzer ver. 3.0 (Atto & Rise Corporation, Tokyo, Japan) and https://www.selleckchem.com/products/AZD1480.html fluorescent intensity of each spot was calculated. Immunohistochemical staining for SH3GL1 protein Immunohistochemistry with the polyclonal antibody against SH3GL1 (sc-25495; Santa Cruz) was performed using commercially available reagents, Histofine (Nichirei Bioscience Inc, Vasopressin Receptor Tokyo, Japan), and according to the manufacturer’s recommendations. This antibody was confirmed to be cross-reactive for human, mouse, and rat SH3GL1. Sections were counterstained with hematoxylin, then dehydrated and mounted. Staining of tissue specimens was observed in 100 × fields with approximately all fields presenting glioma cells.
The staining intensity in cytosole was classified into 5 groups, absent (−), light partial staining (±), homogeneous light staining (+), partly strong positive staining (++) and homogeneous strong positive staining (+++). Brain Tumor Model, Monitoring of Tumor Size, and Serum Sampling Rat C6 glioma cells and 9 L gliosarcoma cells were originally obtained from ATCC and maintained in Dulbecco’s modified Eagle medium (D-MEM) supplemented with 10% fetal calf serum in a humidified atmosphere of 5% CO2. Male Wister rats for C6 cells and Fisher rats for 9 L cells, weighing between 200 and 240 g (7–8 weeks old) were used. The animals were anesthetized and placed in a stereotaxic apparatus. A burr hole was made at 4 mm posterior to bregma and 3 mm right to midline.