The protein samples from airway epithelial cells (A549 and RPMI2650 cells) were prepared in radio immunoprecipitation assay (RIPA) lysis buffer (5 mM sodium fluoride, 10 μg/mL phenylmethylsulfonylfluoride, and 1 mM sodium vanadate) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). Mouse mucosa tissue protein samples were prepared in T-PER Tissue lysis buffer (78510, Thermo Fisher Scientific, T-PER Tissue lysis Protein Extraction Reagent). All samples were separated on 12% SDS–polyacrylamide gels (SDS-PAGE) and then transferred to PVDF membranes, blocked with 5% w/v nonfat dry milk in PBS containing 0.5% Tween-20. The primary antibodies used were actin (CS-8457; rabbit; monoclonal; 1:1000; Cell Signaling, Danvers, MA, USA), caspase-1 (MA5-32909; mouse; monoclonal; 1:1000; Thermo Fisher Scientific, Waltham, MA, USA), IL-1β (GTX74034; mouse; polyclonal; 1:1000; genetex, USA), IκB-α (CS-4814; mouse; monoclonal; 1:1000; Cell Signaling), lamin B1 (AB65986; rabbit; polyclonal; 1:1000; abcam, United Kingdom), NF-κB-p65 (8242; rabbit; monoclonal; 1:1000; Cell Signaling), NLRP3 (AG-20B-0014; mouse; monoclonal; 1:1000; Adipogen, SA, Switzerland), and tubulin (T8203; mouse; monoclonal; 1:1000; Sigma).
Gtx74034
Manufactured by GeneTex
Sourced in United States
The GTX74034 is a laboratory instrument designed for cell culture applications. It provides precise temperature control and monitoring capabilities to support the growth and maintenance of cell lines in a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Dc protein assay, by Bio-Rad (9 mentions)
β actin, by Cell Signaling Technology (5 mentions)
Ag 20b 0042 c100, by Adipogen (5 mentions)
Ag 25b 0006, by Adipogen (3 mentions)
Adi 905 173 100, by Enzo Life Sciences (2 mentions)
Asc adi 905 173 100, by Enzo Life Sciences (2 mentions)
Hmgcl nbp1 58026, by Novus Biologicals (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
N6789, by Merck Group (2 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Caspase 1, by Thermo Fisher Scientific (1 mentions)
T8203, by Merck Group (1 mentions)
Ma5 32909, by Thermo Fisher Scientific (1 mentions)
Ab65986, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ag 20b 0014, by Adipogen (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Cellytic mt cell lysis reagent, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
18 protocols using gtx74034
1
Protein Extraction and Western Blotting
2
Immunoblotting Analysis of Circadian Clock and Inflammatory Proteins
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Protein lysates were prepared using CelLytic MT Cell Lysis Reagent (Sigma) as described previously14 (link), then subjected to SDS-PAGE, with the Precision Plus Protein Dual Color Standard marker (Bio-Rad) and transferred to PVDF membrane (Bio-Rad). Primary antibodies used were REV-ERBα (mouse monoclonal, clone 4F6; 1:1000; Abgent), or REV-ERBβ (mouse monoclonal, clone D-8; 1:1000; Santa Cruz Biotechnology), or NLRP3 (mouse monoclonal, clone Cryo-2; 1:1000; Adipogen), or IL-1β (rabbit polyclonal, GTX74034; 1:1000; GeneTex), and incubated overnight at 4 °C. β-actin (mouse monoclonal, clone C4; 1:10,000; Millipore) was used as a loading control. Membranes were washed with tris-buffered saline with 0.1% Tween-20 (TBS-T), and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (anti-mouse, 7076S, 1:5000; anti-rabbit, 7074S, 1:5000; Cell Signaling) in 3% skim milk in TBS-T. Proteins were visualized using Clarity Western ECL substrate (Bio-Rad), according to the manufacturer’s specifications. Densitometry quantification was performed using Image J 1.46 (NIH). All original blots are provided in Supplementary Fig. 5 .
3
Protein Extraction and Immunoblot Assay
4
Immunoblot Analysis of BMDM Proteins
5
Western Blot Analysis of Inflammasome Proteins
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Cell lysates and culture supernatants were incubated with cell lysis buffer (20 mM Tris HCl pH 7.4, 200 mM NaCl, 1% NP-40) and denatured in laemmli buffer. Subsequently the protein samples were boiled at 95°C for 10 min and separated by SDS-PAGE. Separated proteins were transferred to PVDF membranes. Blocking, incubation with antibody and washing of the membrane were done in PBS supplemented with 0.05% Tween-20 (v/v) and 3% (w/v) non-fat dry milk. Immunoblots were incubated overnight with primary antibodies against caspase-1 (AG-20B-0042-C100, Adipogen), Nlrp3 (AG-20B-0014-C100, Adipogen), ASC (AG-25B-0006, Adipogen), IL-1β (GTX74034, Genetex), IL-18 (5180R-100, Biovision), IkBα (9242S, Cell Signaling), Phospho-IkBα (2859S, Ser32) (Cell Signaling), β-actin (NB600-501H, Novus Biologicals) and A20 (sc-166692, Santa Cruz Biotechnology). Horseradish peroxidase-conjugated goat anti-mouse (115-035-146, Jackson Immunoresearch Laboratories) or anti-rabbit secondary antibody (111-035-144, Jackson Immunoresearch Laboratories) was used to detect proteins by enhanced chemiluminescence (Thermo Scientific).
6
Western Blotting for Inflammasome Proteins
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Unless otherwise stated, cell lysates and culture supernatants were combined for Western blotting. Protein samples were denatured in Laemmli buffer, boiled at 95°C for 10 min, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. PBS supplemented with 0.05% Tween-20 (vol/vol) and 3% nonfat dry milk (wt/vol) was used for blocking and washing of membranes. Immunoblots were incubated overnight with primary antibodies against caspase-1 (AG-20B-0042-C100; 1:1,000; Adipogen), GSDMD (1:1,000; Genentech; Aglietti et al., 2016 (link)), or IL-1β (GTX74034; 1:3,000; Genetex), followed by HRP-conjugated secondary antibodies raised against mouse, goat, or rabbit (1:5,000). The β-actin–HRP antibody was used at 1:5,000 in PBS 0.1% Tween-20 and 5% nonfat dry milk. All proteins were detected by enhanced chemiluminescence.
7
Western Blot Analysis of Immune Signaling
8
Quantification of Nucb2 and NFκB in Mouse Tissues
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Tissues from WT and Nucb2−/− mice were prepared by grinding with mortar and pestle while kept frozen in liquid nitrogen followed by homogenization with RIPA buffer. Bone marrow derived macrophage cell lysates were prepared using RIPA buffer and immediately snap frozen in liquid nitrogen. Samples were vortexed every 10 min for 1 hr. Samples were centrifuged at 14,000 g for 15 min, the supernatant was collected and the protein concentration was determined using the DC Protein Assay (Bio-RAD). Antibodies to Nucb2 (1:1000, N6789; Sigma), IL-1β (1:500, GTX74034; Genetex), NFκB p65 (1:1,000 8242s, Cell Signaling), phosphorylated-NFκB p65 (1:1000 3033S, Cell Signaling), and β-actin (1:1,000 4967L; Cell Signaling) were used at the dilutions specified by the manufacturer. The immune complexes were visualized by incubation with horseradish peroxidase-conjugated anti-rat (PI31470; Pierce) or anti-rabbit secondary antibody (PI31460, Pierce). Immuno-reactive bands were visualized by enhanced chemiluminescence (PI32209; Pierce). Densitometry analysis was performed using the ImageJ Gel Analysis tool, where gel background was also removed individually for each band.
9
Immunoblot Analysis of BMDM Proteins
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The BMDM cell lysates were prepared using RIPA buffer and immediately snap frozen in liquid nitrogen. Samples were vortexed every 10 mins for 1 hour. Samples were then centrifuged at 14,000g for 15 min, the supernatant was collected and the protein concentration was determined using the DC Protein Assay (Bio-RAD). The immunoblot analysis was performed as described previously12 (link). Antibodies to Caspase 1 (1:250, 4B4.2.1 Genentech), IL-1β (GTX74034, Genetex), Caspase 11(NB120-10454, Clone 17D9, Novus Bio), Scot (12175-1-AP, Proteintech), Asc (ADI-905-173-100, Enzo Life Science), Acetylated Histone H3 ( 06-942, LYS9, EMD Millipore), HMGCL (NBP1-58026, Novus Bio), and β-Actin (4967L, Cell Signaling) were used at dilutions specified by the manufacturer. The immune complexes were visualized by incubation with horseradish peroxidase-conjugated anti-rat (PI31470, Pierce) or anti-rabbit secondary antibody (PI31460). Immunoreactive bands were visualized by enhanced chemiluminescence (PI32209, Pierce). Densitometry analysis was performed using the Image J Gel Analysis tool, where gel background was also removed individually for each band.
10
Immunoblotting of Inflammasome Proteins
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Cells and culture supernatants were incubated in cell lysis buffer (20 mM Tris HCl (pH 7.4), 200 mM NaCl, 1% Nonidet P-40) for 10 min on ice followed by denaturation by boiling in Laemmli buffer for 10 min. Protein samples were resolved by SDS-PAGE electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes by semi-dry blotting. The PVDF membrane blocking, antibody incubation, and washing steps were performed using PBS containing 0.05% Tween 20 (v/v) together with 3% (w/v) non-fat dry milk. The incubation of the membranes with primary antibody was performed overnight at 4°C and the primary antibodies used in this study included: caspase-1 (AG-20B-0042-C100, 1:1,000, Adipogen), IL-1β (GTX74034, 1:3,000, GeneTex) and γ-Tubulin (T6557-100UL, 1:1,000, Sigma Aldrich). HRP-conjugated secondary anti-mouse and anti-rabbit antibodies (111-035-144 and 115-035-146; 1:5,000, Jackson ImmunoResearch Laboratories), followed by ECL (Thermo Scientific) incubation were used for signal detection and visualization.
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