Anti β actin igg hrp

The patient-derived GSCs were well characterized (Table 1) and cultured in NS-A medium (90% NeuroCult NS-A Basal Medium Human plus 10% Human NeuroCult NS-A proliferation Supplements, StemCell Technologies, Vancouver, British Columbia, Canada in 3D sphere as we described before [45 (link)]. Complete medium was supplied with recombinant human epidermal growth factor (R&D System, Minneapolis, MN, USA) and 100 units/mL penicillin-100 μg/mL streptomycin (Life Technologies, Carlsbad, CA, USA). Anti-TUSC3 and anti-MGMT antibodies were obtained from Abcam (Cambridge, United Kingdom); anti-β-actin IgG-HRP was obtained from Santa Cruz Biotech (Dallas, TX, USA) and anti-DNMT1, Goat anti-Rabbit IgG-HRP and Goat anti-mouse IgG-HRP were obtained from CellSignaling Technology (Danvers, MA, USA). Temozolomide (TMZ) was obtained from Sigma (St. Louis, MO, USA). 5-Azacitidine (5-Aza) and Lomeguatrib were obtained from Selleckchem (Houston, TX, USA).

U87 and U251 human glioblastoma cell lines were cultured in DMEM (life technologies, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 units/ml penicillin-100 μg/ml streptomycin (Sigma-Aldrich). The culture was maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Temozolomide (TMZ) and Ly294002 were obtained from Sigma-Aldrich. S3I-1757 was kindly provided by Dr. Said Sebti (Moffitt Cancer Center). PIK-75, TGX221, IC-87114 and AS-605240 were purchased from Cayman Chemical (USA). G418 sulfate (50 mg/ml) was obtained from Life Technologies (USA). Stat-3 siRNA was obtained from Sigma-Aldrich (USA). Rabbit anti-Human-Akt, Rabbit anti-Human phospho-Akt, Goat anti-Rabbit IgG-HRP, anti- β-actin IgG-HRP and Rabbit anti-Human cIAP2 were obtained from Santa Cruz Biotech (USA).

40 μg of heat-denatured proteins were loaded on 4–15% precast polyacrylamide gel (Bio-Rad). The proteins were then transferred to PVDF membranes (Bio-Rad), which were blocked with 5% non-fat milk solutions for 1 hour at room temperature. The target proteins were then detected by the primary antibody at 4 °C overnight, washed with 0.1% Tween-PBS and incubated with appropriate secondary antibody for 2 hours. The membranes were then washed and the target proteins were detected with luminol reagent and X-ray film (Santa Cruz). Quantification of the target protein was done using Adobe Photoshop. In brief, the background of the target protein and β-actin were subtracted. Then, the relative expression of the target protein was normalized to β-actin and compared to that of the control group in each experiment. Anti-human HIF-1α antibody was from Novus-bio. Rabbit anti-human BIRC3, goat anti-Rabbit IgG-HRP and anti-β-actin IgG-HRP were obtained from Santa Cruz Biotech.