Ecl western blotting detection kit

Cells (2 × 10⁴ cells/well) were pre-incubated with glucose (5.5 or 30 mM) in a 96-well plate for 48 h and then incubated with 0, 1, 5, 10, or 20 µM HM-chromanone for 48 h. Cell lysates were prepared using ice-cold lysis buffer containing 250 mM NaCl, 25 mM Tris–HCl (pH 7.5), 1% (v/v) NP-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (10 μg/mL aprotinin, 1 μg/mL leupeptin). Cell lysates were washed by centrifugation, and the protein concentrations were determined using a BCATM protein assay kit (Bio-Rad, Hercules, CA, USA). The lysates (30 μg protein) were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels, and the separated proteins were transferred onto nitrocellulose membranes. The membranes were incubated separately with antibodies against Bax, Bcl-2, cytochrome c, caspase 9, caspase 3, and β-actin in TTBS (25 mM Tris–HCl, 137 mM NaCl, 0.1% Tween 20, pH 7.4) containing 5% skim milk for 2 h. The membranes were then washed with TTBS and incubated with secondary antibodies. Signals from secondary antibodies were detected using the enhanced chemiluminescence (ECL) western blotting detection kit (Bio-Rad) and the image was captured using X-ray films. Relative protein expression was quantified by densitometric means using Multi Gauge v3.1 (FujiFilm, Tokyo, Japan) and calculated according to the reference β-actin bands.

Western blots of spinal cord dorsal horn sections with TLR4 (1: 1000, Proteintech, Rosemont, IL, USA, 19188-1-AP), MyD88 (1:1000 Proteintech, 23230-1-AP), p-NF-κB (1:1000, cell signaling, 3033), NF-κB (1:1000, cell signaling, 8482) and TRPV1 (1: 1000; Novus Biologicals, Centennial, CO, USA, NBP1-97417) were produced. The protein bands in the blot were visualized using the ECL Western Blotting Detection Kit and Bio-Rad ChemiDoc XRS system. The band intensity was quantified and plotted using Quantity One Software (Version 4.6.8, Bio-Rad, Hercules, CA, USA).

Hippocampal tissues were homogenized in RIPA buffer with a protease and phosphatase inhibitor cocktail. Cell lysates were centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatants were collected. The protein concentration was determined using a BCA Protein Assay Kit. Protein samples (20 μg) were loaded and electrophoresed by 8–10% SDS-PAGE at 80 V for 120 min. The separated proteins were transferred to PVDF membranes at 1.0 A for 30 min. For blocking, the membranes were incubated with 5% nonfat dry milk in TBS containing 0.1% Tween-20 (TBST) for 1 h at RT. After washing with TBST, the membrane was incubated with primary antibodies at 4 °C overnight. The membrane was then washed with TBST thrice and incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at RT. After washing with TBST thrice, the membranes were detected using the ECL Western blotting detection kit (Bio-Rad, Mississauga, ON, Canada). For protein normalization, β-actin was used as a control. The relative intensities of each band were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Reovirus treated and untreated cultured cells were collected, washed in PBS, and lysed in 150 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA and 1% Nonidet P-40 supplemented with a mixture of protease inhibitors (Roche, Basel, Switzerland). 400 micrograms total protein was pre-cleared and used to immune precipitate TLR3 protein using protein Agarose beads and TLR3 antibody (Antibody online # ABIN201783) for 24 hours at 4°C. The Agarose beads were washed thrice, eluted by boiling with Lameli buffer (Biorad 4x Laemmli Buffer #1610747) and run on a 12% SDS-PAGE. After blotting, membranes were probed with anti-TLR3 primary antibody (Antibody online # ABIN201783). Secondary antibodies were HRP-labeled, and detection was performed using the Clarity Chemiluminescent substrate (Bio-Rad # 1705061). Western blots were performed using standard procedures. Membranes were blocked with 5% milk in TBS containing 0.1% Tween 20, and incubated with antibodies specific for TLR3. Immunoreactive bands were visualized by chemiluminescence (# 1705061 Bio-Rad) clarity ECL western blotting detection kit.

Protein extraction and gel-blot analysis were performed as described previously [35 (link)]. The samples were blotted onto PVDF membranes after SDS—PAGE electrophoresis, and the blots were incubated with anti-PsbD (PSII; AS06 146) and anti-RbcL (Rubisco; AS03 037 primary antibodies (Agrisera, Vännäs, Sweden) overnight at 4 °C followed by the secondary antibody (anti-rabbit IgG horse radish peroxidases conjugated from Agrisera, AS09 602) for 1 h at room temperature according to the manufacturer’s instructions. Signals from immunoblotting were detected using the ECL method (ECL Western Blotting Detection Kit, Bio-Rad, Hercules, CA, USA) by the Invitrogen iBright Imaging Systems (Thermo Fisher Scientific, Pittsburg, PA, USA).

The total proteins of the tissues and cells were extracted by using the RIPA lysis buffer purchased from Beyotime Biotechnology (Shanghai, China) according to the manufacturer’s protocol. The BCA kit (Beyotime Biotechnology, China) was used to quantify the protein concentration. After that, the target proteins were separated by conduction the electrophoresis with 10% SDS/polyacrylamide gel and transferred to the polyvinylidene fluoride (PVDF) membranes produced by Millipore (MA, U.S.A.). The 5% skim milk was diluted by TBS containing 0.1% Tween-20 and incubated with the membranes for 1 h at 37°C. The PVDF membranes were incubated overnight at 4°C with the primary rabbit antibodies against human TGF-β2 (1:1000, ab36495, Abcam, U.K.), Smad2 (1:2000, ab63576, Abcam, U.K.), Smad3 (1:2000, ab28379, Abcam, U.K.), Bax (1:1000, ab32503, Abcam, U.K.), Bcl-2 (1:1000, ab59348, Abcam, U.K.), cleaved caspase-3 (1:1000, ab2302, Abcam, U.K.), VEGF (1:1000, ab39638, Abcam, U.K.), MMP2 (1:1000, ab92536, Abcam, U.K.), MMP9 (1:1000, ab38898, Abcam, U.K.). The horseradish peroxidase-linked goat anti-rabbit IgG (1:2000, ab205718, Abcam, U.S.A.) was incubated with the membranes for 1 h at room temperature. The ECL Western blotting detection kit (Bio-Rad, U.S.A.) was employed to detect the optical density of the protein bands to evaluate the expression levels of the proteins.