Ecl chemiluminescence reagent

EA.hy926 cells were cultured in 6-well cell-culture plates at 200,000 cells per well. The cells were lysed in buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromophenol blue). Equal amounts of protein were separated onto 12% SDS polyacrylamide gels and then transferred to PVDF membranes (Amersham, USA). Membranes were probed with antibodies against p-38, p-p38, IkBα, p-IkBα, p65, p-p65, H3 histone (Cell Signaling), and tubulin (Sigma Aldrich, USA). The blots were developed with appropriate secondary antibody conjugated to HRP (Sigma Aldrich, USA). The membranes were treated with HRP-conjugated secondary antibody (Sigma) and developed with ECL chemiluminescence reagents (Amersham) according to the manufacturer's protocol. The ImageJ software was used for densitometric analysis of the bands.

The protein concentrations of the samples were measured using a BCA protein assay kit (Beyotime Biotechnology, China). The samples containing equal amounts of proteins (30 μg) were denatured in sample loading buffer and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by a transfer to a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences). Then membranes were blocked with 5% (w/v) non-fat dried milk in a Tris-buffered saline with Tween 20 (TBST), and incubated with primary antibodies (anti-Nrf2 (1:500 dilution in TBST; Rabbit, OmnimAbs Co., H-300) and anti-CBR1 (1:500 dilution in TBST; rabbit, Affinity Co., DF7346)) overnight at 4°C. Then membranes were washed three times and incubated with horseradish peroxidase-conjugated anti-goat (for Nrf2) or anti-rabbit (for CBR1) second antibodies (ab6789, Abcam, USA) for 2 h. Immunoreactive bands were visualized using ECL^® chemiluminescence reagents (Amersham Biosciences)according to the manufacturer's instructions, followed by exposure to X-ray films (Sigma Z370398). Films were analyzed with Quantity One Software (BioRad Laboratories), and the resulting absorbance values were expressed as the percentage variation of the control group values. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control.

Control and VHSV-exposed RBCs cell pellets were resuspended in 30 µl of PBS with a cocktail of protease inhibitors (Sigma-Aldrich). Cells were then frozen/thawed 3 times and protein concentration adjusted before loading. Samples were loaded in Tris–Glycine sodium dodecyl sulfate 17% polyacrylamide gels under reducing conditions. Electrophoresis was performed at 100 V for 90 min. For blotting, proteins in the gel were transferred for 75 min at 100 V in transfer buffer (2.5 mM Tris, 9 mM glycine, 20% methanol) to nitrocellulose membranes (BioRad, Madrid, Spain). Membranes were then blocked with 8% dry milk, 1% Tween-20 in PBS and incubated with rabbit polyclonal antibody against human eIF2α-P (36.1 KDa) or rabbit polyclonal antibody against human α-Actin (42 KDa,) in PBS containing 0.5% dry milk, and 0.5% Tween-20 (PMT buffer), overnight at 4°C. Membranes were then washed 3 times with PMT buffer for 15 min before incubation with GAR-Po (Sigma-Aldrich) in PMT buffer for 45 min. Finally, membranes were washed 3 times with PBS containing 0.5% Tween-20. Peroxidase activity was detected using ECL chemiluminescence reagents (Amersham Biosciences, Buckinghamshire, UK) and revealed by exposure to X-ray. Protein bands were analyzed by densitometry using the Scion Image 4.0.2 Software (RRID: SCR_008673) (
www.scionorg.com).

Proteins of cells were obtained according to this method [20 (link)]. The untreated or treated LoVo cells were washed three times with ice-cold PBS, and the protein was extracted using a RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). The protein concentration was detected using a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, China), and 25 μg protein was separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (Sigma-Aldrich, St Louis, MI, USA) for 1 h at room temperature, the membranes were incubated with primary antibodies including anti-DDR1, anti-PI3K, anti-p-AKT, anti-AKT, anti-MDM2, anti-P53, anti-BAX, anti-Bcl-2, and anti-β-actin (1:1,000) at 4°C overnight. Next, the membranes were washed with Tris-buffered saline containing 0.05% Tween-20 (Sigma-Aldrich) thrice and incubated with peroxidase-conjugated secondary antibodies (1:3,000) for 2 h at room temperature. Finally, the membranes were developed using ECL chemiluminescence reagents (Amersham Pharmacia Biotech, Tokyo, Japan). The protein band intensities were determined using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Melatonin (N-acetyl-5-methoxytryptamine), soybean oil, Bradford reagent, PMSF, BSA, propidium iodide, protease inhibitor cocktail, anti-p53, anti-Bax, anti-Bcl-x,
HRP-conjugate and formalin were procured from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. EGTA, tris-HCL, trichloroacetic acid, tween-20, tween-100, skimmed milk powder, and phosphate buffer saline (PBS) were purchased from HiMedia, Mumbai, India. EDTA, NaCl ABTS
(2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate), and ethanol were from Merck, Germany. ECL chemiluminescence reagent was from Amersham Pharmacia Biotech, Piscataway, NJ, USA. Dimethyl sulfoxide
(DMSO) and sodium dodecyle sulphate were from Calbiochem, San Diego CA USA. DNase free RNase was procured from Genei, Bangalore, India.

Worms were collected from the plates with M9 buffer, transferred to tubes, centrifuged and washed three times to eliminate bacteria. The final worm pellet was resuspended in lysis buffer (25 mM HEPES, 10 mM Na₄P₂O₇ 10H₂O, 100 mM NaF, 5 mM EDTA, 2 mM Na₃VO₄, 1% Triton X-100) containing 1% protease inhibitor (PI) cocktail (Sigma-Aldrich). The samples were sonicated 3 times on ice for 10 seconds and centrifuged for 15 minutes at 12,000 rpm and 4°C to eliminate the cuticles. Cells were washed with PBS and lysed in lysis buffer. Worms and cell protein lysates were subjected to SDS-PAGE on 10% polyacrylamide gel. Proteins were electroblotted to PVDF membrane (Millipore Corp, Bedford, MA). Blots were reacted with specific primary antibodies and with respective HRP-conjugated secondary antibodies obtained from the Jackson ImmunoResearch Laboratories. SuperSignal WestPico chemiluminescence substrate (Thermo Scientific Inc., Waltham, MA) and ECL chemiluminescence reagent were used (Amersham Pharmacia Biotech, UK) for visualizing the blots.