[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Next, to analyze the expression of mRNA and GPNMB protein in MG, M, and neuronal cells, we performed Northern and WB analyses (Fig. fragments, but type 2 MG and M did not, although all these cells expressed mRNA and the full\length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and M, may have furin\like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double\immunstaining with 9F5 antibody and anti\Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist mutant K-Ras G12C-IN-3 mice [Gpnmb^tm1(Gfp)Mcf] were generated as described in Supporting Information. The day after overnight mating was considered embryonic day 0 (E0), and the day of birth was designated postnatal day 0 (P0). Animals were treated according to the Guidelines of the Institutional Animal Care and Use Committee of Kumamoto University and UNITECK and the Animal Care Committee of Niigata University of Pharmacy Mouse monoclonal to AKT2 and Applied Life Sciences. Cell Culture Primary type 1 and type 2 MG were harvested from primary mixed glial cell cultures prepared from neonatal Wistar rat pups as previously reported (Sawada et al., 1990; Shimizu et al., 2008; Suzumura et al., 1987). Type 1 MG were stimulated with lipopolysaccharide (LPS; 0.1 g?ml?1; serotype 0127:B8; SigmaCAldrich) for 24 hr, and type 2 MG were stimulated with rat IL\4 (5 ng?ml?1; PeproTech EC) for 96 hr. These MG were stored at ?80C until use. Primary rat neurons, astrocytes, and neuroepithelial cells were prepared and cultured as described previously (Nakashima et al., 1999; Tajiri et al., 2004). Primary rat peritoneal M and thioglycolate\elicited M were obtained as described previously (Woo et al., 1990). The murine MG cell lines Ra2, 6\3, and MG5 were maintained as described previously (Kanzawa et al., 2000; Ohsawa et al., 1997). Monkey kidney COS\7 cells and human HEK293 cells were cultured in Dulbecco’s altered Eagle’s medium supplemented with 10% fetal bovine serum. Rat primary type 1 MG was treated with rat recombinant IL\12 (10 K-Ras G12C-IN-3 ng?ml?1; R&D Systems). Establishment of Mouse Hybridoma Clones Female BALB/c mice (6 weeks aged, cDNA clone was isolated via RT\PCR with total RNA from rat type 1 MG. PCR was carried out with rat primers corresponding to nucleotides 53\1860 (GenBank, accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”BC061725″,”term_id”:”38197643″,”term_text”:”BC061725″BC061725). The product obtained was inserted into pGEM\T easy vector (Promega Corp.) to yield pGEM\T\rcDNA was released by were used (Tsuji et al., 2002). The cDNA and cDNA were subcloned into the (21 oligonucleotides; Pesu et al., 2006) was obtained from SigmaCAldrich. COS\7 cells were transfected with plasmids via Effectene Transfection Reagent (QIAGEN) according to the manufacturer’s protocol. HEK293 cells were cotransfected with plasmids and siRNA by using TransMessenger Transfection Reagent (QIAGEN). As a negative control, the same amount of insert\less plasmid was transfected. For the immunostaining assay, at 72 hr after transfection, cells were fixed in 4% paraformaldehyde in PBS for 10 min and treated with PBS made up of 0.05% Triton X\100. Cells were then incubated with 9F5 (1 g?ml?1) and goat anti\GPNMB antibody (AF2330, 1 K-Ras G12C-IN-3 g?ml?1) and then with Alexa Fluor488\ or 594\labeled antibodies against mouse or goat immunoglobulins (1:500; Molecular Probes). Fluorescence intensities were quantified as described previously (Kawahara et al., 2012). After photomicrographs were imported into the Scion Image system (NIH), they were quantified via NIH ImageJ software (National Institutes of Health; http://rsb.info.nih.gov/nih-image/). A manually set threshold intensity was kept constant, and the number of pixels in 9F5\ and anti\GPNMB antibody\immunostained cells in an area measuring 400 400 m2 was decided. At least three areas were quantified per one transfection. The total data for three or four experiments were expressed as means??SEM. RNA Blot Analysis The total RNA from cells and rat brain were prepared by using the Isogen reagent (Nippon Gene) according to the manufacturer’s recommendations. After electrophoresis through formaldehyde\made up of agarose gels, RNAs were transferred to nylon membranes. Digoxigenin\labeled antisense RNA probes were synthesized, via.