NuPage® 4–12% polyacrylamide gels, LDS sample buffer, See-Blue Plus2 protein markers, and TRIzol® reagent were purchased from Invitrogen (Carlsbad, CA, USA); iScript™ cDNA synthesis Kit and SYBR™ green supermix from Bio-Rad Laboratories (Hercules, CA, USA), primers for real-time PCR from Integrated DNA Technologies (Coralville, IA, USA). Antibodies against Claudin-1 (which also recognizes Claudin-3) and -2 were purchased from ThermoFisher Scientific (Waltham, MA, USA). Goat polyclonal actin primary antibody, and horseradish peroxidase-conjugated secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and SuperSignal West Dura substrate from Pierce (Rockford, IL, USA). A mouse TH1/TH2 9-Plex assay kit was purchased from MesoScale Discovery (Gaithersburg, MD, USA). All other reagents used were commercially available and were of the highest quality available.
Nupage 4 12 polyacrylamide gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPAGE 4%-12% polyacrylamide gels are laboratory consumables used for protein separation and analysis via gel electrophoresis. They provide a stable and consistent matrix for the separation of proteins based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti mdm2, by Abcam (2 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (2 mentions)
Rabbit anti β actin, by Merck Group (2 mentions)
Vinculin, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Seeblue plus 2 protein markers, by Thermo Fisher Scientific (1 mentions)
Antibodies against claudin 1, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Sybr green supermix, by Bio-Rad (1 mentions)
Anti lamin a c, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
12 protocols using nupage 4 12 polyacrylamide gel
1
Protein Expression Analysis in Cell Lines
2
Whole Cell Protein Expression Analysis
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Whole cell protein extracts (lysis buffer: 30 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM KCl, 2 mM EDTA, 5% Glycerol, 0.1% Triton X-100, and 1× Protease Inhibitor Cocktail—Sigma-Aldrich, St. Louis, MO) were quantified by BCA assay (Pierce, Rockford, IL), separated onto NuPAGE 4–12% polyacrylamide gels (Invitrogen, Carlsbad, CA) and blotted on nitrocellulose membranes (Amersham, England). Membranes were hybridized with polyclonal antibodies anti-PARP-1 (#9542 Cell Signaling Technology, MA), anti-Caspase-3 (#9665 Cell Signaling Technology), anti-PUMA/BBC3 (#4976 Cell Signaling Technology, MA), anti-HSP90 (#4877 Cell Signaling Technology, MA), anti-Lamin-A/C (#sc-6215 Santa Cruz Biotechnology), anti-p21 (#sc-756, Santa Cruz Biotechnology), anti-TP53INP1(#NBP1-76638 Novus-Biologicals) or monoclonal antibodies anti-Bcl-2 (#sc-509 Santa Cruz Biotechnology), anti-BTG2 (#SAB1404558 Sigma-Aldrich), and anti-p53 (#sc-126 Santa Cruz Biotechnology). Monoclonal antibody either anti-Actin (#CP01, Calbiochem) or anti-α-Tubulin (#T5168 Sigma-Aldrich) was used as a loading control. Bands were visualized and quantified with FluorChem E System (Protein Simple, CA) or with ChemiDoc XRS+ and ImageLabTM software (Bio-Rad Laboratories, Inc, Hercules, CA).
3
Protein Extraction and Western Blot Analysis
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Cells were washed in ice-cold PBS and lysed with radio-immunoprecipitation buffer (20 mM Tris-HCl, pH = 7.4, 100 mM NaCl, 1% NP-40, 0.1% (w/v) sodium dodecyl sulphate, 1% (w/v) sodium deoxycholate, 50 mM NaF, 50 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol and cOmplete™ Protease Inhibitor Cocktail (Roche Applied Science)) for 10 min on ice. Lysates were cleared using QIAshredder spin columns (Qiagen), boiled in Laemmli buffer and resolved on NuPAGE™ 4–12% polyacrylamide gels (Invitrogen). Proteins were transferred onto a nitrocellulose membrane and stained using the following antibodies: goat anti-MxB (Santa Cruz Biotechnology, sc-47197, 1:500), rabbit anti-MxA (Novus Biologicals, H00004599-D01P, 1:1000), mouse anti-HSV-1/2 VP16 monoclonal antibody LP175 (link) (kindly donated by A. Minson and H. Browne, University of Cambridge, Cambridge, UK, 1:5000), rabbit anti-GAPDH (Santa Cruz Biotechnology, sc-25778, 1:3000) and mouse anti-GST (Santa Cruz Biotechnology, sc-57753, 1:1000). Densitometric analyses were performed using MultiGauge version 3.0. Values were normalised to the loading control and are represented relative to the appropriate control condition. Uncropped scans of all immunoblots can be found in Supplementary Figs. 8 –12 .
4
Protein Expression Analysis via Western Blot
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Proteins were lysed and extracted through incubation with radioimmunoprecipitation assay (RIPA) buffer (Sigma Aldrich). Bradford reagent was used to quantify the extracted proteins. About 40 μg of protein were separated by Nupage 4%–12% polyacrylamide gels (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Aurora, OH, USA). The membranes were blocked with 5% milk in phosphate-buffered saline with 0.05% Tween 20 (PBS-T) buffer and then incubated with the following primary antibodies: mouse anti-MDM2 (1:1000, Abcam, Cambridge, UK), p85 (1:1000 Cell Signaling), p53 (1:2500, Abcam, Cambridge, UK), p21 (Santa Cruz Biotechnology, TX, USA 1:1000), and rabbit anti-α-actin (1:2500, Sigma Aldrich), at 4 °C overnight. The membrane was washed with PBMS-T and then incubated for 1 h, at room temperature, with goat anti-rabbit or goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000, GE Healthcare). The membranes were developed using a chemiluminescence reaction (ECL, GE Healthcare). The data were analyzed using ImageJ software (NIH, Bethesda, MD, USA).
5
SARS-CoV-2 RBD Protein Detection
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Cell lysates were obtained by diluting cell pellets in NuPAGE™ LDS Sample Buffer (4X) (Thermo Fisher Scientific, Cat# NP0007) and incubating at 99°C for 5 minutes, separated by electrophoresis in NuPAGE 4-12% polyacrylamide gels (Thermo Fisher Scientific, Cat#NP0321PK2) and then transferred to PVDF membranes. The membranes were probed with an anti-SARS-CoV-2 RBD rabbit polyclonal antibody (Sino Biological Inc., 40592-T62) followed by a goat anti-rabbit HRP-conjugated secondary antibody (Thermo Fisher Scientific, Cat# A16110, RRID AB_2534782 ). Membranes were developed by chemiluminescence using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Cat# WBKLS0500) and recorded using ChemiDoc MP Imaging System (Bio-Rad). An anti-GAPDH antibody (Cell Signaling Technology Cat# 2118, RRID:AB_561053 ) was used as loading control.
6
Protein Extraction and Western Blot
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To extract proteins, cells were lysed in RIPA lysis buffer (Sigma), supplemented with proteases (A32955, ThermoFisher Scientific) and phosphatases inhibitors (1862495, ThermoFisher Scientific). Proteins were separated on NuPAGE 4–12% polyacrylamide gels (ThermoFisher Scientific) and then transferred onto nitrocellulose membranes. Membranes were probed with the appropriate primary (anti-GHITM, 16296-1-AP, Proteintech; anti-Actin, ab8226, Abcam) and secondary antibodies (31430 and 31460, Invitrogen) and visualised with SuperSignal West Pico Substrate (34080, ThermoFisher Scientific). Images were analysed with ImageJ (Fiji).
7
Western Blot Protein Analysis Protocol
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Protein extraction was performed using RIPA buffer (Sigma-Aldrich, City of Saint Louis, MO, USA) and total lysate was quantified by Bradford reagent. Twenty-five micrograms of protein was separated on Nupage 4–12% polyacrylamide gels (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to polyvinylidene difluoride membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked with 5% milk in phosphate-buffered saline with 0.05% Tween 20 (PBS-T) buffer to be probed with the following antibodies: Anti-PI3K p85 (mAb4292, Cell Signaling, Danvers, MA, USA), Phospho-Akt (Ser473) (mAb4060, Cell Signaling, Danvers, MA, USA), Akt (pan) (mAb4691, Cell Signaling, Danvers, MA, USA), and rabbit anti-β-actin (Sigma-Aldrich, City of Saint Louis, MO, USA), by standard protocol. For detection, goat anti-rabbit or goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (GE Healthcare, Chicago, IL, USA) were used accordingly. Signal detection was performed via chemiluminescence reaction (GE Healthcare, Chicago, IL, USA).
8
SARS-CoV-2 RBD Protein Expression Analysis
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5 × 104 (link) HuH7 cell/well were seeded in 12-well plates and incubated overnight at 37°C. On the following day, cells were pre-incubated for 2 h with adenovirus 5 (Ad5) at an MOI of 20 PFU/cell, and infected with an MOI of 5 × 105 of AC1 or AC3. Cells were harvested 72 h later and lysed with NuPAGE LDS Sample Buffer (4×) (Thermo Fisher Scientific, catalog no. NP0007) at 99°C for 5 min. Proteins were separated by electrophoresis in NuPAGE 4%–12% polyacrylamide gels (Thermo Fisher Scientific, catalog no. NP0321PK2) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were probed with an anti-SARS-CoV-2 RBD rabbit polyclonal antibody (Sino Biological, 40592-T62) followed by a goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Fisher Scientific, catalog no. A16110, RRID: AB_2534782 ). Membranes were developed by chemiluminescence using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, catalog no. WBKLS0500) and recorded using ChemiDoc MP Imaging System (Bio-Rad). An anti-GAPDH antibody (Cell Signaling Technology, catalog no. 2118, RRID: AB_561053 ) was used as loading control.
9
Quantitative Western Blot Analysis
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Cells were lysed by radioimmunoprecipitation buffer and proteins were then extracted and quantified by Bradford reagent. An amount of 50 mg of total proteins were loaded on Nupage 4%–12% polyacrylamide gels (Thermo Fisher Scientific, Waltham, MA, USA) for each sample and transferred to polyvinylidene difluoride membranes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA), then they were probed with the following antibodies separately: mouse anti-p53 (1:1,000, Cat No: ab28, Abcam, Cambridge, UK) and rabbit anti-β-actin (1:2,000, Cat No: ab3280, Abcam). For detection, goat antirabbit or goat antimouse secondary antibodies conjugated to horseradish peroxidase (1:2,000, GE Healthcare Bio-Sciences Corp.) were used. Protein abundance was measured by chemiluminescence reaction (ECL, GE Healthcare Bio-Sciences Corp.). Western blot and quantification were performed using ImageJ software analysis (National Institutes of Health, Bethesda, MD, USA).
10
Western Blot Analysis of MDM2 and p21
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Proteins were extracted by incubation with RIPA buffer and quantified by Bradford reagent. Twenty-five micrograms of protein were separated on Nupage 4–12% polyacrylamide gels (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride membranes (PVDF, GE Healthcare Life Science, Piscataway, NJ, USA) to be probed with the following antibodies: mouse anti-MDM2 (1 : 500, Abcam, Cambridge, UK); mouse anti-p21 (1 : 1000, Cell Signaling, Danvers, MA, USA), and rabbit anti-β-actin (1 : 5000, Sigma-Aldrich). For detection, goat anti-rabbit or goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (1 : 2000, GE Healthcare Life Science) were used. Signal detection was performed via chemiluminescence reaction (ECL, GE Healthcare Life Science). WB quantification was performed using ImageJ software analysis (National Institutes of Health, Bethesda, MD, USA).
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