Most chemicals used in this study were purchased from Sigma–Aldrich (St. Louis, MO, USA) including the followings: glucose, palmitate, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoilium bromide (MTT), DL-fluorocitric acid barium salt, Oil Red O and Eosin Y. Nile Red and Hematoxylin. BODIPY™-palmitate (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-caspase 3, anti-phospho-AKT, anti-total AKT, anti-phospho-GSK3b, anti-total GSK3b, anti-phospho-JNK, anti-total JNK, anti-phospho-P65, and anti-total P65 antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-actin and anti-tubulin antibodies were purchased from Bethyl Laboratory (Montgomery, TX, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. The catalog numbers of all reagents and antibodies were listed in Supplementary Table 2 of SI .
Anti total p65
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-total P65 is a primary antibody that recognizes the p65 subunit of the NF-kB transcription factor. It can be used to detect and quantify the total p65 protein levels in cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho p65, by Cell Signaling Technology (3 mentions)
Dl fluorocitric acid barium salt, by Merck Group (1 mentions)
Anti phospho gsk3b, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti total jnk, by Cell Signaling Technology (1 mentions)
Anti total akt, by Cell Signaling Technology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Palmitate, by Merck Group (1 mentions)
Nitrocellulose, by Bio-Rad (1 mentions)
Antibodiesdetecting cleaved parp and caspase 3, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti c myc, by Abcam (1 mentions)
Lps rs, by InvivoGen (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Pam2csk4, by InvivoGen (1 mentions)
Anti ikkβ, by Cell Signaling Technology (1 mentions)
Anti phospho ikkα β, by Cell Signaling Technology (1 mentions)
Cryo 2, by Adipogen (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
4 protocols using anti total p65
1
Lipid Metabolism Regulation Assay
2
Protein Expression Analysis in Pleural Tumors
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Total protein lysates were fractionated on 4%-15% polyacrylamide gels and transferred onto nitrocellulose (Bio-Rad). These primary antibodies were used: anti-MTDH at 1:500 dilution (2F11C3 monoclonal antibody, Thermo Fisher), anti–phospho-p65 at 1:1000 with anti–total p65 at 1:1000 (Cell Signaling Technologies), anti–c-Myc at 1:500 (Abcam), and anti–β-Actin (Sigma-Aldrich). Additionally, antibodies detecting cleaved PARP and caspase-3 (Cell Signaling) were used to assess apoptosis activity. Subsets of samples were randomly selected from the original group of 41 tumors and 14 normal pleurae for this series of experiments. All protein experiments were performed in triplicate. To avoid problems related to incomplete membrane stripping, separate blots were used for each independent experiment.
3
SARS-CoV-2 Protein Interaction Assay
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PAM2CSK4, PAM3CSK4, LPS-RS and recombinant nucleocapsid protein fused to His-tag were purchased from InvivoGen. Recombinant soluble E protein from SARS-CoV-2 fused to GST (E-GST) was purchased from Clinisciences. GST, GST-Nef and the corresponding antibodies were produced in our laboratory. Soluble recombinant TLR2 and spike protein fused to His-tag were purchased from R&D systems. Anti-spike rabbit polyclonal antibodies were purchased from Invitrogen. Anti-TLR2 and anti-TLR4 monoclonal antibodies were obtained from eBioscience. Anti-phospho-P65 and anti-total P65 were purchased from Cell Signalling. Bay11-7082, SB202190, PD98059 and RO318220 were purchased from Calbiochem. The MyD88 inhibitor ST 2825 P65 was purchased from MedChemExpress.
4
Western Blot Analysis of NLRP3 and NF-κB Signaling
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Cell lysates were collected in radioimmunoprecipitation buffer (150 mmol/L NaCl, 1% IGEPAL, 0.5% deoxycholic acid, 0.1% SDS, 50 mmol/L Tris, pH 7.5) containing protease inhibitor cocktail (Complete, mini, EDTA-free; Roche). Samples were separated on 8% SDS-PAGE gels then transferred to nitrocellulose membranes (Amersham Biosciences).
Membranes were blocked with 5% skim milk and probed with anti-NLRP3 antibody (Cryo-2; Adipogen), anti-phospho-p65, anti-total p65, anti-phospho-IKKα/β, anti-IKKβ, anti-phospho-IRAK4, anti-total IRAK4 or anti-β-actin (all from Cell Signaling). Bound primary antibodies were detected with HRP-conjugated anti-rabbit (Dako). Labelled proteins were visualized by incubating the membrane in ECL Prime reagent (Amersham Biosciences) and using an ImageQuant LAS 4000 (GE Healthcare). Sodium fluoride was added to radioimmunoprecipitation buffer and skim milk at 20 mM or to other buffers at 2 mM when assessing phosphorylation. Densitometry was performed using ImageJ.
Membranes were blocked with 5% skim milk and probed with anti-NLRP3 antibody (Cryo-2; Adipogen), anti-phospho-p65, anti-total p65, anti-phospho-IKKα/β, anti-IKKβ, anti-phospho-IRAK4, anti-total IRAK4 or anti-β-actin (all from Cell Signaling). Bound primary antibodies were detected with HRP-conjugated anti-rabbit (Dako). Labelled proteins were visualized by incubating the membrane in ECL Prime reagent (Amersham Biosciences) and using an ImageQuant LAS 4000 (GE Healthcare). Sodium fluoride was added to radioimmunoprecipitation buffer and skim milk at 20 mM or to other buffers at 2 mM when assessing phosphorylation. Densitometry was performed using ImageJ.
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