Rabbit polyclonal antibody to β actin

CeA tissue was homogenized in RIPA lysis buffer containing protease inhibitors and centrifuged at 16,000×g for 20 min at 4 °C. The total protein level in the supernatant was determined with the bicinchoninic acid assay, and the proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis before being transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were first incubated with the following primary antibodies: rabbit polyclonal antibody to CRF (Abcam), rabbit polyclonal antibody to CRFR1 (Abcam), rabbit polyclonal antibody to NOP (Abcam), or rabbit polyclonal antibody to β-actin (Abcam). The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The specific protein bands were visualized using enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK), and their densities were analyzed with ImageJ software (NIH, Bethesda, MD, USA).

The protein levels of F98 IDH1 –WT/–R132H, IDH2 –WT/–R172K, and Mock gliomas were evaluated by immunoblot analysis. Cells were lysed in ice–cold lysis buffer composed of 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β–glycerophosphate, 1 mM Na₃VO₄, 1 μg/mL leupeptin, and protease inhibitor cocktail (Sigma, MO, USA), and the concentration of lysate protein was evaluated with the bicinchoninic acid method (Pierce Biotechnology, Rockford, IL, USA). Approximately 50 μg of protein was loaded in each lane of a polyacrylamide denaturing gel for electrophoresis. After electrophoresis, the protein was transferred to nitrocellulose membranes for blotting. We used a rat monoclonal antibody to IDH1 (Dianova, Hamburg, Germany), a mouse monoclonal antibody to IDH1–R132H (Dianova), a rabbit polyclonal antibody to IDH2 (Proteintech, Chicago, IL, USA), a mouse monoclonal antibody to IDH2–R172K (NewEast Biosciences, King of Prussia, PA, USA), and a rabbit polyclonal antibody to β–actin (Abcam, Cambridge, UK). Primary antibodies were detected by horseradish peroxidase–conjugated antibodies (Santa Cruz Biotechnology, Paso Pobles, CA, USA).

The antibody against mouse IL-1β was a gift from R. Solari (Imperial College, London), whereas the antibody against mouse caspase-1 was a gift from P. Vandenabeele (Ghent University, Belgium)64 (link)65 (link). Other antibodies used were monoclonal anti-tubulin from Sigma-Aldrich; rabbit polyclonal antibody to β-actin from Abcam; anti-IL-1α (ALF161) from ebioscience.