Anti cleaved caspase 3 ab32042 antibody

Protein specimens were diluted in loading buffer and denatured at 95°C. The total protein (40 μg) was electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto the polyvinylidene difluoride membrane. Five percent skimmed milk was used to block the membrane at room temperature for 2 h. The membrane was incubated with the primary antibodies anti-CYLD, anti-Bax, anti-Bcl-2, anti-cleaved-caspase 3, and anti-GAPDH (Abcam, USA) overnight at 4°C, followed by washing and incubation with corresponding secondary antibodies for 2  h. At length, protein samples were subjected to an enhanced chemiluminescence detection system (Bio-Rad, USA). anti-CYLD antibody (ab137524), anti-Bax antibody (ab263897), anti-Bcl-2 (ab196495), anti-cleaved-caspase 3 antibody (ab32042) and anti-GAPDH antibody (ab8245) were purchased from Abcam (Shanghai, China).

Primary hepatocytes were cultured in 8-well chamber slides and immunocytochemistry was performed on them. Primary hepatocytes were fixed in 4 % paraformaldehyde and incubated with primary monoclonal antibodies: Anti-Cleaved Caspase-3 antibody (ab32042, abcam, Cambridge, UK), diluted in PBS/1%, BSA/0.3%, Triton^TM X-100 (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C, and a Texas-red-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch) diluted in PBS/1%, BSA/0.3%, Triton^TM X-100 overnight at 4°C for 1 h. Nuclei were stained with DAPI (Sigma-Aldrich). Images were captured with the BZ-X710 fluorescence microscope, and the ratio of Caspase 3-positive cells per image was analyzed using ImageJ (NIH).

Geraniol and D-gal were purchased from Sigma Aldrich in Germany. Antibodies against HO-1 (# PA5-77833), PARP (PA5-16452), pAkt (# 44-621G), NQO1 (# PA5-82294), pPI3K (#PA5-104853), BCL2 (# PA5-27094), PI3K (# PA5-29220), Nrf2 (# PA5-105664), β-actin (# PA5-78716) and Akt (# 44-609G) were obtained from Invitrogen; Thermo Fisher Scientific, Inc., (Waltham, MA, USA). Anti-cleaved caspase-3 (ab32042) antibody was purchased from Abcam (Branford, CT, USA). MDA (Cat no: 700870), SOD (Cat. no: 706002), GPx (Cat. no: 703102), and CAT (Cat. no: 707002) were measured with kits (from Cayman Chemical, Ann Arbor, MI, USA) and all experiments were done according to the manufacturer’s protocol.