Cells were harvested in cell lysis buffer (50 mM NaCl, 50 mM TRIS pH8, 1% IGEPAL, 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma) and allowed to incubate on ice for 20 mins. Lysates were then centrifuged (16,000 × g for 20 min at 4°C) and the supernatant collected. Lysates were mock or Endo H (NEB) digested according to the manufacturer’s instructions for 90 mins at 37°C as required. Lysates were denatured by heating at 95°C for 5 mins in reducing sample buffer (Bio-Rad) and resolved by SDS-PAGE on precast polyacrylamide gels (Biorad) before immunoblotting onto PVDF membranes. Membranes were probed with the designated primary antibodies in 3% BSA in PBST, followed by incubation with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (all Santa Cruz Biotechnology). The following primary antibodies were utilized: anti-MR1 CT (McWilliam et al., 2016 (link)), anti-MR1 and anti-HLA-A, B, C (Abcam), anti-GFP, anti-GAPDH, anti-gD, and anti-ICP0 (all Santa Cruz Biotechnology).
Reducing sample buffer
Manufactured by Bio-Rad
Sourced in Italy
Reducing sample buffer is a laboratory reagent used to prepare samples for protein analysis. It contains reducing agents that help denature and solubilize proteins, making them more accessible for analysis. The core function of this product is to prepare samples for downstream applications such as gel electrophoresis and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (5 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Precast polyacrylamide gel, by Bio-Rad (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Endo h, by New England Biolabs (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Anti icp0, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Anti mr1 ct, by Abcam (2 mentions)
Anti gd, by Santa Cruz Biotechnology (2 mentions)
Anti gfp, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (2 mentions)
Image multi gauge software, by Fujifilm (2 mentions)
Las 4000 imaging system, by Fujifilm (2 mentions)
Gradient polyacrylamide gel, by Bio-Rad (2 mentions)
Pro il 1β, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer system, by Santa Cruz Biotechnology (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Mouse anti gapdh antibody, by Merck Group (1 mentions)
Ab24509, by Abcam (1 mentions)
12 protocols using reducing sample buffer
1
MR1 and HLA-A,B,C Protein Detection
2
Quantification of Liver Protein Expression
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About 50 mg of frozen mouse liver tissue samples were homogenised in 650 µL of TNE buffer (10 mmol/L Tris-HCl (pH 7.5), 1% Nonidet P40, 150 mmol/L NaCl, 1 mmol/L EDTA) containing a cocktail of protease and phosphatase inhibitors (Roche). Lysates were incubated at 4°C for 45 min and then centrifuged at 12 000 g for 5 min. Protein content was quantified in the supernatant using BCA Protein Assay (Thermo Fisher). Lysates were mixed with the reducing sample buffer (Bio-Rad) for electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes (Thermo Fisher). Equal loading (50 µg) was verified using Ponceau red solution. Membranes were incubated with anti-PPARα (1:1000, Ab24509, Abcam) antibody. After secondary antibody incubation (anti-rabbit, 1:3000; Amersham, GE Healthcare, Courtaboeuf, France), immunodetection proceeded using an enhanced chemiluminescence kit (Clarity Western ECl Substrate, BioRad, France) and bands were revealed using the Las-4000 Imaging System and Image MultiGauge software (Fujifilm, Tokyo, Japan). After initial immunodetection, membranes were stripped of antibodies and reprobed with mouse anti-GAPDH antibody (1:20 000; Millipore) and secondary antibody (anti-mouse, 1:3000; Amersham GE healthcare).
3
SDS-PAGE and Immunoblotting of Venoms
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Electrophoresis of venoms in sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels was carried out with 20% gels using Tris-Glycine as running buffer and stained with either Coomassie Brilliant Blue R-250 or silver staining, as outlined in [20 (link)]. For immunoblotting, venom samples (typically 10–15 μg protein) were solubilized in reducing sample buffer (BioRad), separated by SDS-PAGE, and subsequently transferred to nitrocellulose paper. Membranes were blocked with blocking buffer [1% (w/v) skimmed non-fat milk, 0.3% (v/v) Tween 20/PBS] for 1 h and then incubated with therapeutic sera from immunized horses, diluted in the same blocking buffer (1/1,000), for 1.5 h. Membranes were washed with 0.05% (v/v) Tween 20/PBS and incubated with goat anti-horse horseradish peroxidase-conjugated secondary antibody (Sigma) (diluted 1/50,000) in blocking buffer for 1 h. Membranes were washed once again and blots were developed using Luminata Forte Western HRP Substrate (Millipore).
4
Western Blot Analysis of Macrophages
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Macrophages (106 per condition) were treated as indicated, washed once with ice cold PBS, and lysed with RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP–40, 0.5% sodium deoxycholate, 0.1% SDS) with addition of a protease and protein phosphatase inhibitor cocktail (Pierce, Rockford, IL). Lineage-depleted bone marrow cells and FACS-isolated bone marrow monocytes and granulocytes were lysed as above. Samples were boiled in reducing sample buffer (Bio-Rad) and run on SDS-PAGE gels (Bio-Rad), transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and blocked in 5% nonfat dry milk in PBS–0.1% Tween 20. Primary antibodies included anti-phospho-ERK1/2 (clone D13.14.4E), anti-ERK1/2 (clone 137F5), and anti-β-actin (clone D6A8) (all from Cell Signaling Technology, Danvers, MA). Primary antibodies were incubated in blocking buffer overnight at 4°C; membranes were then washed three times in PBS–0.1% Tween 20, treated with secondary antibody (horseradish peroxidase-linked goat anti-rabbit IgG, catalog number 7074, Cell Signaling Technology) in blocking buffer, washed three times, and treated with enhanced chemiluminescence reagent (Pierce). Membranes were exposed to film (Amersham GE Healthcare, Pittsburgh, PA), and images were scanned and analyzed. Densitometric analysis was performed using ImageJ software [17 (link)].
5
NR4A1 Protein Interactome Profiling
6
Quantifying TSLP and JAK/STAT Pathway in Mouse Liver
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Frozen mouse liver tissue samples (about 50 mg) were homogenized in 650 mL of TNE buffer (1 mmol/L EDTA, 1% Nonidet P40, 10 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl) containing a phosphatase inhibitors and cocktail of protease (Roche, Basel, Switzerland). The incubation condition was 4°C for 45 min and the centrifugation condition was at 12 000 g for 5 min. BCA Protein Assay (Thermo Fisher, MA, USA) was conducted to quantified protein content was quantified in the supernatant. Reducing sample buffer (Bio-Rad) was used to mix lysates for electrophoresis and subsequently the lysates were transferred onto polyvinylidene fluoride membranes (Thermo Fisher). Equal loading (50 mg) was verified using Ponceau red solution. Antibodies against TSLP (ab188766) was purchased from Abcam (MA, USA) and p-JAK1 (#3331S), JAK1 (#50994S), p-STAT1 (#9167S), STAT1 (#14994S), p-STAT3 (#52075S), STAT3 (#9139S) and anti-β-actin (#3700S) were obtained from Cell Signaling Technology (Danvers, MA, USA). After incubating with HRP-labeled goat anti-rabbit IgG (H+L) (1:3000; Beyotime, Shanghai, China), the Clarity Western ECl Substrate (BioRad, CA, USA) was conducted to proceed immunodetection and the Las-4000 Imaging System and Image MultiGauge software (Fujifilm, Tokyo, Japan) were used to reveal bands.
7
Purification and Analysis of Lung CD4+ T Cells
8
MR1 and HLA-A,B,C Protein Detection
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Cells were harvested in cell lysis buffer (50 mM NaCl, 50 mM TRIS pH8, 1% IGEPAL, 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma) and allowed to incubate on ice for 20 mins. Lysates were then centrifuged (16,000 × g for 20 min at 4°C) and the supernatant collected. Lysates were mock or Endo H (NEB) digested according to the manufacturer’s instructions for 90 mins at 37°C as required. Lysates were denatured by heating at 95°C for 5 mins in reducing sample buffer (Bio-Rad) and resolved by SDS-PAGE on precast polyacrylamide gels (Biorad) before immunoblotting onto PVDF membranes. Membranes were probed with the designated primary antibodies in 3% BSA in PBST, followed by incubation with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (all Santa Cruz Biotechnology). The following primary antibodies were utilized: anti-MR1 CT (McWilliam et al., 2016 (link)), anti-MR1 and anti-HLA-A, B, C (Abcam), anti-GFP, anti-GAPDH, anti-gD, and anti-ICP0 (all Santa Cruz Biotechnology).
9
Western Blot Analysis of Tumor Cells
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Tumor cells were plated (5.105 cells/dish) in 10-cm dishes, for 72 h. After this time, adherent cells were harvested and lysed in RIPA buffer; protein preparations (30-40 μg) were resuspended in reducing Sample buffer (Bio-Rad Laboratories, Segrate, Mi, Italy) and heated at 95°C for 5 min. Following electrophoretic separation by SDS-PAGE, proteins were transferred onto nitrocellulose membrane. After blocking, membranes were incubated with the primary antibodies against HDAC-6, SMAD7, α-actin and α-tubulin. Detection was done using horseradish peroxidaseconjugated secondary antibodies, and enhanced chemiluminescence ECL-Prime reagents. Total extracts were normalized by using an anti α-tubulin antibody.
10
MR1 Protein Immunoblotting and Endo H Digestion
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Cells were harvested in cell lysis buffer (50 mM NaCl, 50 mM TRIS pH8, 1% IGEPAL, 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma) and allowed to incubate on ice for 20 min. Lysates were then centrifuged (16,000 × g for 20 min at 4°C) and the supernatant collected. Lysates were mock or Endo H (NEB) digested according to the manufacturer’s instructions for 90 min at 37°C as required. Lysates were denatured by heating at 95°C for 5 min in reducing sample buffer (Bio-Rad) and resolved by SDS- PAGE on precast polyacrylamide gels (Bio-Rad) before immunoblotting onto PVDF membranes. Membranes were probed with the designated primary antibodies in 3% BSA in PBST, followed by incubation with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (all Santa Cruz Biotechnology) and visualised using Clarity Western ECL Substrate (Bio-Rad). The following primary antibodies were utilised: anti-MR1 CT,51 (link) anti-MR1 (Abcam) and anti-HLA-A, B, C (Abcam), anti-GFP (Santa Cruz Biotechnology), anti-GAPDH (Santa Cruz Biotechnology or ThermoFisher), anti-DDDDK (anti FLAG, Abcam) and anti-ICP27 (Santa Cruz Biotechnology). Immunoblots depicted in Figures 2 C and 3 B are from the same experiment and depict the same Mock and RAd-Ctrl lanes. Unrelated samples were cropped from Figure 3 B.
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