Ecl detection system

For each sample, all the cells were lysed for 30 min in lyses buffer on 4°C; after ultrasound, the cell debris was centrifuged at 12,000 rpm for 20 min at 4°C, protein concentration was detected by BCA (Beyotime, Haimen, China), using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the proteins, the separated proteins were blotted onto PVDF membrane, blocking 2 h at room temperature with 5% nonfat dry milk in TBST, incubating the antibody for 16 h at 4°C with 1:1,000 concentration, washing PVDF membrane 3 times for 10 min in TBST, incubating secondary antibody at 25°C for 2 h with the 1:10,000 concentration, washing membranes 3 times for 10 min in TBST. Finally, the membranes were visualized with the enhanced chemiluminescence (ECL) detection system (GE Healthcare, Piscataway, NJ, USA). All of the primary antibodies (p-JNK, JNK, P62, LC3-II, GAPDH) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cells were lysed in SDS sample buffer, separated by SDS-PAGE, and detected by immunoblotting analysis with the indicated antibodies. Antibodies were visualized using the ECL detection system (GE Healthcare).

Cells were lysed in TNE buffer with complete mini protease inhibitor (Roche). The 10 µg of protein samples were separated on a SDS-polyacrylamide gel. The primary antibodies for phosphorylated p65 and p65 (Cell Signaling) and GAPDH (Merck) were purchased. Polyclonal anti-NOXO1 antibody was generated (Scrum, Tokyo, Japan). The ECL detection system (GE Healthcare, Buckinghamshire, UK) was used for detection.

Proteins were extracted from cells with a lysis buffer containing 50 mM Tris (pH 7.5), 5 mM EDTA, 150 mM NaCl, 0.25% Triton X-100 and 1 mM DTT. Protease inhibitors (Complete protease inhibitor cocktail, Roche) and phosphatase inhibitors (50 mM NaF, 1 mM Na3VO4) were added to the basic buffer. Twenty microgram proteins were electrophoresed on a SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. After a one-hour blocking in Tris buffered saline Tween 0.1% (TBST) containing 5% milk, membranes were incubated overnight at 4 °C with mouse monoclonal RhoA (Santa Cruz, sc-418, 1:1000), rabbit polyclonal RhoB (Santa Cruz, sc-180, 1:1000) and tubulin (Santa Cruz, 1:500) primary antibodies. Membranes were then washed three times in TBST and incubated for one hour with horseradish peroxidase-conjugated secondary antibody (1:2000). Detection was then performed with the ECL detection system (GE Healthcare).

Whole cell extracts from cultured cells were prepared using radioimmunoprecipitation assay buffer (Thermo Scientific, Rockford, IL) containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein quantification was done using a BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions. Total protein (20 μg) was loaded onto 4–12% bis–tris gels with 3-(N-morpholino) propanesulfonic acid buffer and separated by a NuPAGE electrophoresis system (Invitrogen). Protein was transferred to Invitrogen™ polyvinylidene difluoride and immunoblotting was carried out according to standard protocols. Monoclonal antibody against PMS2 was purchased from Origene Technologies (Rockville, MD) and monoclonal antibody against GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) was used to confirm equal loading. The membrane was washed and then incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology). Protein complexes were visualized with the Echo-chemiluminescence (ECL) Detection System (GE Healthcare, Little Chalfont, UK) using the Chemidoc Imaging System (Bio-Rad Laboratories, Hercules, CA).

Harvested liver tissues and cells were lysed with mammalian lysis buffer containing phosphatase and protease inhibitors. Equal amounts of cell lysates were measured with the BCA protein assay reagent (Pierce Biotechnology, Rockford, IL). Lysates were boiled in sample buffer containing β-mercaptoethanol for 5 min. Proteins were then subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (GE healthcare). After blocking with 5% skim milk in PBS, membranes were incubated with appropriate dilutions of antibodies at 4°C overnight as follows: HO-1 (Enzo, ADI-OSA-150, 1 : 1000 dilution), PGC-1α (Abcam, ab72230, 1 : 1000 dilution), COX III (Abcam, ab110252, 1 : 20000 dilution), COX IV (Cell Signaling, #4844S, 1 : 1000 dilution), p-eIF2α (Cell Signaling, #9721S, 1 : 1000 dilution), and β-actin (Cell Signaling, #4967S, 1 : 2000 dilution). Membranes were then washed with 0.05% PBS-Tween 20 and incubated with a 1/5000 dilution of HRP-conjugated secondary Abs at room temperature for 1 h. Immunoreactivity was detected by using the ECL detection system (GE Healthcare). Films were exposed at multiple time points to ensure that the images were not saturated. The relative band density was analyzed by using ImageJ software (US National Institutes of Health, Bethesda, MD).

20 µg of whole cell lysates or nuclear extracts of 1 × 10⁶ cells were separated by SDS-PAGE and subsequently transferred onto nitrocellulose membranes (Whatman, Dassel, Germany). The blots were probed with antibodies against bcl-x, cytochrome c, caspase-3, XIAP (all purchased from BD Biosciences, Heidelberg, Germany), ERK 1/2, mcl-1, survivin, Rel-A, Rel-B (all purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, USA), p38, pp38 (all purchased from Cell Signaling Technology^®, MA, USA). GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, USA) was used as a loading control. Signals were detected using the ECL detection system (GE Healthcare, Munich, Germany).