Anti bax

Protein extraction and western blot analysis were performed as previously described [39 (link)].
Cells were harvested for protein extraction 48h after transfection. Lysates from transfected cells were collected using RIPA buffer (Sigma R0278, St Louis, MO, USA). Protein lysates were separated on SDS-polyacrylamide gels, electroblotted onto polyvinylidene difluoride membrane and immunoblotted with primary antibodies, then with peroxidase-conjugated secondary antibodies. After three additional washes, the immune complex was detected by ECL detection (Thermo 34077; Rockford, IL, USA). The following antibodies were used: anti-Cyclin A2 (1540-1, Epitomics, Burlingame, CA, USA), anti-Cyclin B1(1495-1, Epitomics), anti-Cyclin D1(1677-1, Epitomics), anti-Cyclin E(4129;Cell Signaling Technology, Beverly, MA, USA), anti-Bcl-2 (1017-1, Epitomics), anti-PUMA(1652-1, Epitomics), anti-mdm2 (556353, BD PharMingen, San Diego, CA, USA), anti-Caspase-3 (1087-1, Epitomics), anti-p21(2990-1, Epitomics), anti-PARP-1 (1072-1, Epitomics), anti-Bax (1063-1, Epitomics), anti-β-actin (60008-1-Ig, Protein Tech, Chicago, IL, USA), goat anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotechnology, Dallas, Texas) and goat anti-rabbit IgG-HRP (sc-2004, Santa Cruz Biotechnology).

Histopathological analysis and immunohistochemistry were carried out as described [18] (link). Anti-p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1∶200), anti-p-IKKα/β (Cell Signaling Technology, Danvers, MA, USA; 1∶100), anti-p-S6(S235/236) (Cell Signaling Technology; 1∶400), anti-AKT (Abcam, Cambridge, MA, USA; 1∶100), anti-p-AKT(S473) (Abcam; 1∶100); anti-Bax (Epitomics, Burlingame, CA, USA; 1∶300), anti-Ki67 (Bioss, Beijing, China; 1∶50), anti-IL-1β (Abzoom, Dallas, USA; 1∶200), anti-IL-6 (Sanying, Wuhan, China; 1∶300), and anti-TNF-α (Boster, Wuhan, China, 1∶150) were used in the immunohistochemical studies. Secondary antibody was diluted 1∶100 and applied for 2 h at room temperature. The Vectastain ABC Elite System (Vector Laboratories, Burlingame, CA) was used to visualize staining for IHC. The immunostained slides were observed under a microscope. Images were taken by a digital camera and semi-quantity of the signal was analyzed by counting mean density of the immunoreactivities against all primary antibodies. Brown or yellow was regarded as positive signal. Image data were analyzed with NIS-Elements AR 3.0 software (Nikon, Japan). Immune cell, apoptosis, and proliferation indices were generated by counting the number of positive cells per high-powered field (HPF; 40× objective) within each mouse [19] (link).

Two stable clones of BGC823-Gastrin KD cells were selected and, respectively, named as PshrNa-A1and PshrNa-B3. BGC823-Ctrl cells and untreated BGC823 cells were taken as control. These cells were subjected to lysis (50 mmol/L Tris–Cl (pH 6.8), 100 mmol/L DTT, 2% SDS, and 10% glycerol). Equal amounts of protein (50 μg) were then separated on 12% SDS–PAGE and transferred to PVDF membrane (Pharmacia, (GE), USA). The membrane was blocked with 5% nonfat milk for 1 h and then incubated with the specific antibody: anti-gastrin (Abcam, Cambridge, MA, USA) (1 : 500), anti-IκB-α (Santa Cruz, CA, USA) (1 : 500), anti-p-IκB-α (Santa Cruz, CA, USA) (1 : 500), anti-NF-κB (Santa Cruz, CA, USA) (1 : 500), anti-Bcl-2 (US Biological, Boston, USA) (1 : 500), anti-Bax (Epitomics, Burlingame, CA, USA) (1 : 500), anti-COX17(Abcam, Cambridge, MA, USA) (1 : 500), anti-COX5B(Abcam, Cambridge, MA, USA) (1 : 500), anti-ATP5J(Abcam, Cambridge, MA, USA) (1 : 500), and anti-β-actin (Santa Cruz, CA, USA) (1 : 20000) overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, CA, USA) were used at a 1 : 2000 concentration. Color development was performed with the ECL system (GE Health Care, Little Chalfont, Buckinghamshire, UK).

Total proteins from H9c2 cells and working heart tissues were extracted using RIPA lysis buffer, separated by electrophoresis on an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MS, USA). The membrane was blocked with 5% nonfat milk in Tris-buffered saline/Tween 20 (TBST) and was incubated overnight with the following primary antibodies: anti-ox-CaMKIIδ (Met281/282) (1 : 1000, GeneTex), anti-p-CaMKIIδ (Thr286) (1 : 1000, Abcam), anti-CaMKIIα (1 : 10000, Abcam), anti-CaMKIIβ (1 : 500, Abcam), anti-CaMKIIδ (1 : 1000, Abcam), anti-CaMKIIγ (1 : 500, Abcam), anti-p-JNK1/2 (T183/Y185) (1 : 1000, CST), anti-JNK1/2 (1 : 1000, CST), anti-p-NF-κB p65 (Ser536) (1 : 1000, Abcam), anti-NF-κB p65 (1 : 1000, Abcam), anti-Bcl-2 (1 : 500, Abcam), anti-Bax (1 : 500, Abcam), anti-cytochrome c (1 : 1000, Abcam), and anti-GAPDH (1 : 1000, ZSGB-Bio, Beijing, China). The membrane was then washed and probed with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody (1 : 8000, ZSGB-Bio) to enhance the chemiluminescence that was detected using a Fusion-FX6 imaging system (Vilber Lourmat, Marne-la-Valle, France).

Phosphate-buffered saline (PBS, pH = 7.4) was purchased from Sangon Co, Ltd (China), 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), polyacrylamide, bis-acrylamide, and TARMA were purchased from Sigma-Aldrich Co, Ltd (USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin solution were purchased from Invitrogen. Other chemicals were purchased from Sigma-Aldrich Co, Ltd. Anti-Bcl-2, anti-Bax, and anti-Caspase-3 proteins were purchased from Abcam Co. Ltd (Great Britain).