Bcl2 associated x (bax)

Western blotting was performed as described previously 17 (link). The primary antibodies against acetylated histone H3, acetylated histone H4, and β-actin were purchased from Santa Cruz Biotechnology (CA, USA). The primary antibodies against p21, p27, cdc25C, cyclin B1, Bim, Bax, Bcl-2, Bcl-xL, RAGE, and LC3 were purchased from Epitomics (CA, USA). The primary antibodies against p-cdc25C, cdc2, p-cdc2, Cyto-C, cleaved caspase-9, cleaved caspase-3, cleaved caspase-7, cleaved-PARP, PARP, PI3KC3, Beclin 1, and p62 were purchased from Cell Signaling Technology (MA, USA). All secondary antibodies were purchased from Jackson Immuno Research Laboratories (PA, USA).

The levels of the pro-apoptotic proteins (Bax (BioVision, Inc. (San Francisco, CA, USA) Catalog # E4513) and caspase-3 (Sigma-Aldrich (Saint-Louis, MO, USA) Catalog # CASP3C-1KT)) and the anti-apoptotic protein (Bcl-2, Cusabio (Wuhan, China) Catalog # CSB-E08854r) in the cortical homogenate were estimated using commercial kits according to the manufacturer’s protocols.

Total proteins from TBP/Q₇₉-GFP SH-SY5Y cells were obtained using a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and a protease inhibitor cocktail (Sigma-Aldrich). After quantitation using a protein assay kit (Bio-Rad, Hercules, CA, USA), proteins (20 μg) were separated on 10−12% SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes (Sigma-Aldrich) by reverse electrophoresis. After blocking, the membrane was probed with CREB (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), pCREB (S133) (1 : 1000; Santa Cruz Biotechnology), BCL2 (1 : 500; BioVision, Milpitas, CA, USA), GADD45B (1 : 1000; Abcam, Cambridge, MA, USA), BAX (1 : 500; BioVision), NRF2 (1 : 500; Santa Cruz Biotechnology), AMPKα (1 : 1000; Cell Signaling, Danvers, MA, USA), pAMPKα (T172) (1 : 1000; Cell Signaling), GAPDH (1 : 1000) (MDBio Inc., Taipei, Taiwan), or β-actin (1 : 5000; Millipore, Billerica, MA, USA) primary antibody at 4°C overnight. The immune complexes were detected using a horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG antibody (1 : 10000; GeneTex, Irvive, CA, USA) and a chemiluminescent substrate (Millipore).

Caspase 9 and caspase 3 activities were evaluated by the colorimetric kits (BioVision Inc., Milpitas, CA, USA). The cells were mixed with cell lysis buffer and centrifuged at 10,000× g for 1 min. Proteins were quantitated, added to the reaction buffer and chromophore DEVD-pNA (aspartyl-glutamyl-valyl-aspartyl-p-nitroanilide) at 37 °C for 2 h, and these activities were finally measured at 400 nm. The relative enzyme activity was expressed as % of the control group.
Apoptotic regulators such as Bax, Bcl-2, p-p38, p38, p-JNK, and JNK (BioVision Inc.) were isolated by 10% SDS-PAGE and determined by Western blotting mentioned above. The transferred proteins on the nitrocellulose membrane were identified by the corresponding primary antibodies (Santa Cruz Biotechnology Inc.) and incubated with the secondary antibodies and chemiluminescence reagent (Opti-ECL HRP Reagent Kit, Bioman Scientific Co., Ltd.). Protein expression of β-actin was an internal control. The membrane was stripped by the method described above for incubation with different primary antibodies. The image of protein bands was examined by the method described above.

Frozen left ventricles were homogenized in a cocktail of protease inhibitors (Complete ®, Roche Diagnostics, Indianapolis, IN, USA) by means of a polytron (PT-MR2100, Kinematica, Luzern, Switzerland), and protein content was determined by BCA assay (Pierce BCA protein assay kit, Thermo Scientific, Rockford, IL, USA). Eighty micrograms of protein were loaded on acrylamide gel and electrophoretically separated. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (0.45 µm Millipore, Billerica, MA, USA) and incubated overnight with polyclonal primary antibodies provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA): ICAM-1, VCAM-1, MMP-2 and -9, IkB, pIkB, NF-kB, iNOS,14-3-3ε, Bad, pBad, and β-actin; Lamin A from Abcam (Cambridge, MA, USA), Bax from BioVision (Mountain View, CA, USA), and Bcl-2 from Invitrogen (Rockford, IL, USA). After washing with PBS, membranes were incubated with appropriate horseradish peroxidase-coupled secondary antibodies. Chemiluminisence was developed using an Immobilion Chemiluminescent Millipore kit (Billerica, MA, USA). Images from films were digitally acquired by means of a digital camera and analyzed using the calibrated densitometer GS-800 USB (Bio-Rad Laboratories, Hercules, CA, USA). β-actin or Lamin A were used as load control [21 (link),41 (link)].