Anti cd133 w6b3c1

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing 1% NP-40, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl. After sonication and centrifugation of the lysates, proteins (20 µg) were subjected to SDS-PAGE in 7.5% poly-acrylamide gels, followed by immunoblot analysis. The following antibodies were used: anti-BRG1 (a gift from Dr. Tsutomu Ohta, National Cancer Center, Japan), anti-NS (A300–600A; Bethyl Laboratories, Montgomery, TX, USA), anti-GAPDH (3H12; Medical & Biological Laboratories (MBL), Nagoya, Japan), anti-CD133 (W6B3C1; Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-CD44 (2C5; R&D Systems, Minneapolis, MN, USA). Signals were detected by LAS-3000 (Fujifilm, Tokyo, Japan), quantified with ImageJ software (National Institutes of Health, USA) and normalized using GAPDH loading control.

CEP-1347 was purchased from TOCRIS Bioscience (Bristol, UK) and was dissolved in DMSO to prepare a 1 mM stock solution. Anti-CD133 (W6B3C1) was from Miltenyi Biotech (Bergisch Gladbach, Germany). Antibodies against Sox2 (#3579), Bmi1 (#6964), GFAP (#3670), and GAPDH (#5174) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA), except that the antibodies used for the detection of Sox2 (MAB2018) and Bmi1 (05-637) in glioma stem cells were purchased from R&D Systems Inc. (Minneapolis, MN, USA) and Millipore (Billerica, MA, USA), respectively. Ant-E-cadherin (sc-8426) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies and Alexa Fluor^® 488-conjugated secondary antibody were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA) and Thermo Fisher Scientific, (Waltham, MA, USA), respectively.

Domatinostat (4SC-202; S7555) was purchased from Selleck Chemicals (Houston, TX, USA). Domatinostat was dissolved in DMSO to prepare a 10 mM stock solution. The structure of domatinostat was obtained from DrugBank [38 (link)]. Propidium iodide (P3566) and Hoechst33342 (H3570) solutions were purchased from Thermo Fisher Scientific, (Waltham, MA, USA). Trypan blue solution (T8154) was purchased form Merck (Darmstadt, Germany). Antibodies against Bmi1 (05-637) and Nestin (MAB5326) were purchased from Merck. An antibody against SOX2 (MAB2018) was purchased from R&D Systems Inc. (Minneapolis, MN, USA). Anti-CD133 (W6B3C1) was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany). Antibodies against, OCT-4a (#2890), GAPDH (#5174), cleaved PARP (#9541), and cleaved caspase 3 (#9661) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

Antibodies against Sox2 (#3579), Nanog (#4903), Bmi1 (#6964), GFAP (#3670), phospho-c-Jun (#9261), c-Jun (#9265), and GAPDH (#5174) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-CD133 (W6B3C1) was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany). The antibodies used to detect the expression of Sox2 (MAB2018) and Bmi1 (05-637) in GS-Y01 cells were purchased from R&D Systems Inc. (Minneapolis, MN, USA) and Millipore (Billerica, MA, USA), respectively. AS602801 was purchased from ChemScene (Monmouth Junction, NJ, USA) and was dissolved in DMSO to prepare a 10 mM stock solution.

Antibodies against PLCε (07-513), PLCγ1 (05-366), Bmi1 (05-637), and Nestin (MAB5326) were purchased from Merck Millipore (Billerica, MA, USA). An antibody against SOX2 (MAB2018) was purchased from R&D Systems Inc. (Minneapolis, MN, USA). Antibodies against GFAP (#3670), Nanog (#4903), phospho-JNK (#4668), phosphor-c-Jun (#2361), OCT-4A (#2890), GAPDH (#5174), were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Antibodies against PLCβ1 (sc-205), PLCδ3 (sc-514912), and GFP (sc-9996) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-PLCδ1 (610356) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-CD133 (W6B3C1) was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany). U73122 (U6756) was purchased from Merck Millipore.

Cellular populations derived from magnetic separation were grown onto glass slides, fixed with freshly prepared 4% paraformaldehyde, washed in PBS and reacted with the anti-PLC-β2 (#SC-206, Santa Cruz Biotechnology, Santa Cruz, CA) antibody in a Net Gel solution, alone or in combination with the anti-CD133 (W6B3C1, Miltenyi Biotech) or with the anti-EpCAM (NCL-ESA, Leica Biosystems, Buccinasco, I) antibodies, respectively, following a previously reported procedure [18 ]. Samples were then incubated with a FITC and/or TRITC conjugated secondary antibody and, after washes, with 0.5 mg/ml 4',6-diamidino-2-phenylindole (DAPI), dried with ethanol and mounted in glycerol containing 1,4-diazabicyclo [2.2.2] octane (DABCO) to retard fading. Fluorescent samples were observed with a Nikon Eclipse TE2000-E microscope (Nikon), acquiring cell images by the ACT-1 software for a DXM1200F digital camera (Nikon S.p.a., Florence, I). To measure PLC-β2 staining, digitized images were analyzed with the ImageJ software, following the manufacturer’s instructions (http://rsb.info.nih.gov/ij/).