HFF cells (5×104 cells per well) were infected with virus (m.o.i. 1) and treated with the concentrations of compounds indicated in the text or the equivalent volume of DMSO. All samples were prepared for western blotting by washing cells once in PBS, then suspending cells directly in 50 µl 2× Laemmli buffer containing 5 % β-mercaptoethanol before incubating at 95 °C for 5 min. Western blotting of proteins was carried out as described elsewhere [31 (link)], using antibodies recognizing IE1/2, UL57, pp28, (all Virusys, 1 : 1000 dilution), UL86 (a kind gift from Wade Gibson, Johns Hopkins School of Medicine, 1 : 1000 dilution) and β-actin (Sigma, 1 : 5000 dilution). All primary antibodies were incubated overnight at 4 °C and detected using an anti-mouse- or anti-rabbit-HRP-conjugated antibody (New England Biolabs). Chemiluminescence solution (GE Healthcare) was used to detect secondary antibodies on film.
Chemiluminescence solution
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
Chemiluminescence solution is a reagent used in laboratory analysis to detect and quantify specific analytes. It generates light through a chemical reaction, which can be measured and used to determine the presence and concentration of the target analyte in a sample. The core function of this product is to provide a reliable and sensitive method for detecting and quantifying various biological and chemical substances in a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (8 mentions)
β actin, by Merck Group (5 mentions)
Anti mouse or anti rabbit horseradish peroxidase hrp conjugated antibodies, by Merck Group (4 mentions)
Mini protean tetra cell equipment, by Bio-Rad (4 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Thermo Fisher Scientific (4 mentions)
Lumino image analyzer las 3000 mini, by Fujifilm (4 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Rabbit anti nrf2, by Abcam (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Las 4000 lumino imaging unit, by Fujifilm (2 mentions)
Anti human aim2, by Cell Signaling Technology (2 mentions)
Anti human asc, by Cell Signaling Technology (2 mentions)
Pro caspase 1, by Cell Signaling Technology (2 mentions)
Anti human il 1β, by R&D Systems (2 mentions)
Ripa buffer, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
β actin, by Fujifilm (2 mentions)
Clone 5a8, by Merck Group (2 mentions)
Ie2 proteins clone 5a8, by Merck Group (2 mentions)
Rabbit anti bdnf, by Abcam (1 mentions)
15 protocols using chemiluminescence solution
1
Western Blot Analysis of HCMV Proteins
2
Assessing Nrf2 and BDNF Levels via Western Blot
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Nrf2 and BDNF levels were assessed by Western blotting as described in the previous study [16 (link)]. Briefly, animals (n = 5 per group) were sacrificed 24 h after ischemia/reperfusion under deep anesthesia with a mixture of 75 mg/kg alfaxalone and 10 mg/kg xylazine on the 56th day of diet feeding. The left hippocampus was homogenized briefly, and the nuclear and cytosolic fractions were separated using extraction kits following the manufacturer’s instructions (Abcam). Homogenized proteins were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis. Thereafter, the gel was transferred onto a nitrocellulose membrane (Pall Crop, East Hills, NY, USA), and the membrane was incubated with rabbit anti-Nrf2 (1:1000, Abcam) and rabbit anti-BDNF (1:5000, Abcam). Protein bands were visualized using a chemiluminescence solution (GE Healthcare, Buckinghamshire, UK) and were normalized versus laminin A + C and β-actin levels, respectively, as demonstrated in the previous study [20 (link)].
3
Immunoblot Analysis of Inflammatory Pathways
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Cells were harvested and lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease inhibitor cocktail (Sigma-Aldrich). Proteins samples were separated using 10–15% SDS-PAGE and then transferred to membranes (Millipore, Burlington, MA, USA), which were then probed with primary and secondary antibodies. Membranes were developed using a chemiluminescence solution (GE Healthcare, Chicago, IL, USA) in a LAS-4000 Lumino-imaging unit (Fujifilm, Tokyo, Japan). Immunoblot band intensities were quantified using NIH ImageJ software (Version 1.53t, Fujifilm), and results were presented as intensity ratios versus β-actin. The antibodies used were as follows; anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-human iNOS (Santa Cruz Biotechnology), anti-human TLR2 (Santa Cruz Biotechnology), anti-human TLR4 (Santa Cruz Biotechnology), NF-κB (Cell Signaling Technology), MAPK Family antibody sampler kit (Cell Signaling Technology), anti-human AIM2 (Cell Signaling Technology), anti-human ASC (Cell Signaling Technology), pro-Caspase-1 (Cell Signaling Technology), anti-human IL-1β (R&D Systems, Minneapolis, MN, USA), Beclin1 (Santa Cruz Biotechnology), ATG5 (Santa Cruz Biotechnology), and LC3 (Cell Signaling Technology).
4
Western Blot Analysis of HCMV Infection
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Lysates of uninfected or infected HFF cells (see text and figure legends) were prepared for Western blotting by washing cells once in PBS and then suspending them directly in 2× Laemmli buffer containing 5 % β-mercaptoethanol before incubation at 95 °C for 5 min. Western blotting of proteins separated on 8 or 10 % polyacrylamide gels was carried out as described elsewhere [24 (link)], using antibodies recognizing IE1/2, UL44, pp28, (all Virusys, 1 : 1000 dilution), β-actin (SIGMA, 1 : 5000 dilution), PDGFRa (ABCAM ab203491, 1 : 1000 dilution), Hand1 (ABCAM, 1 : 1000 dilution), GATA-3, GATA-4 and HMGA2 (all Cell Signaling Technology, all 1 : 1000 dilution). All primary antibodies were incubated overnight at 4 °C and detected using anti-mouse- or anti-rabbit-horseradish peroxidase (HRP)-conjugated antibodies (Cell Signaling Technology). Chemiluminescence solution (GE Healthcare) was used to detect secondary antibodies on film. Where necessary, blots were stripped and reprobed.
5
Western Blot Analysis of HCMV Proteins
6
Quantitative Western Blot Analysis
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Two hundred thousand cells per well were plated in a 6-well plate and allowed to settle overnight, followed by the treatment with varying doses of the compounds/stressors. Control and treated cells were harvested and washed with PBS (X2), followed by lysis in RIPA buffer (89900, Thermo Fisher Scientific) containing complete protease inhibitor cocktail (4693159001, Roche Applied Science, Penzberg, Bavaria, Germany) on ice for 45 min. Lysates were separated on an SDS-polyacrylamide gel using Mini-Protean® Tetra cell equipment (Bio-Rad, Hercules, CA, USA), and subjected to western blotting using protein-specific antibodies as indicated and horseradish peroxidase-conjugated secondary HRP antibody (31430 or 31460, Thermo Fisher Scientific). Blots were developed using chemiluminescence solution (GE Healthcare, Buckinghamshire, UK) and visualized using a Lumino Image Analyzer (LAS 3000-mini; Fuji Film, Tokyo, Japan). Band intensity was quantified using ImageJ software (NIH) and plotted as a percentage using Microsoft™ Office© 2016. Statistical significance was calculated by an unpaired t-test of GraphPad® software (2018) using mean, SD, and N from four independent experiments, and shown as * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.
7
Nrf2 Protein Expression Analysis
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Nrf2 protein levels were assessed by western blot analysis to observe the effects of PEP-1-PGK1 on Nrf2-mediated antioxidant cascades, as stated previously [32 (link)]. The animals were sacrificed with isoflurane 1 d after ischemia. The hippocampi were dissected, homogenized, and loaded onto a sodium dodecyl sulfate–polyacrylamide gel for electrophoresis. Thereafter, the gel was transferred onto a nitrocellulose membrane (Pall Crop, East Hills, NY, USA), which was incubated with rabbit anti-Nrf2 (1:1000, Abcam) and laminin B (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein bands were visualized using a chemiluminescence solution (GE Healthcare, Buckinghamshire, UK), and Nrf2 protein bands were normalized to those in laminin B.
8
Quantitative Analysis of TMPRSS2 Expression
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A total of 2.5 × 105 cells per well were plated in a 6-well plate, allowed to settle overnight, followed by treatment with each of the withanolides. The control or treated cells were incubated at 37 °C and 5% CO2. After 48 h, control and treated cells were harvested and washed twice with PBS, followed by lysis in RIPA buffer (89,900, Thermo Fisher Scientific) containing complete protease inhibitor cocktail (4,693,159,001, Roche Applied Science, Penzberg, Bavaria, Germany) on ice for 45 min. Lysates were separated on an SDS-polyacrylamide gel using Mini-Protean® Tetra cell equipment (Bio-Rad, Hercules, CA, USA) and subjected to western blotting using protein-specific primary antibodies [anti-TMPRSS2 (AbCam ab92323), anti-β-actin (AbCam ab49900)], and horseradish peroxidase-conjugated secondary HRP antibody (31,430 or 31,460, Thermo Fisher Scientific). Blots were developed using chemiluminescence solution (GE Healthcare, Buckinghamshire, UK) and visualized using a Lumino Image Analyzer (LAS 3000-mini; Fuji Film, Tokyo, Japan). Band intensity was quantified using ImageJ software (NIH) and plotted as a percentage using Microsoft™ Office 2016.
9
Immunoblot Analysis of HCMV Proteins
10
Quantifying Protein Expression by Western Blot
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Cells (2 × 105/well) were plated in 6-well plates and allowed to settle overnight, followed by the treatment with varying doses of resveratrol/stressors. Control and treated cells were harvested and washed with PBS (× 2), followed by lysis in RIPA buffer (Thermo Fisher Scientific, 89900) containing complete protease inhibitor cocktail (Roche, 4693159001) on ice for 45 min. Lysates were separated on an SDS-polyacrylamide gel using Mini-Protean® Tetra cell equipment (Bio-Rad, Hercules, CA, USA), and subjected to western blotting using protein-specific antibodies as indicated and horseradish peroxidase-conjugated secondary HRP antibody (Thermo Fisher Scientific, 31430, 31402 or 31460). Blots were developed using chemiluminescence solution (GE Healthcare, Buckinghamshire, UK) and visualized using a Lumino Image Analyzer (LAS 3000-mini; Fuji Film, Tokyo, Japan). Band intensity was quantified using ImageJ software (NIH, 1.52a) and plotted as a percentage using Microsoft™ Office© 2020. Relative band intensity in control was taken as 100%.
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