Jejunum proteins were prepared using a RIPA lysis kit (Solarbio, Beijing, China). The protein concentration was examined via the bicinchoninic acid (BCA) method. Equivalent amounts of total protein were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes. The membranes were blocked in Tris-buffered saline and Tween 20 (TBST) containing 5% bovine serum albumin (BSA) for 2 h at room temperature and incubated first with primary antibodies against GAPDH (1:10 000, Proteintech, China), TLR4 (1:500, Proteintech, China), MyD88 (1:1000, Proteintech, China), IκB (1:2000, Proteintech, China), p-IκB (1:1000, Cell Signaling Technology, USA), IL-1β (1:1000, Thermo Fisher, USA), IL-6 (1:500, Thermo Fisher, USA), IL-8 (1:600, R&D System, USA), and TNF-α (1:500, R&D System, USA) at 4 ℃ overnight and then with HRP-conjugated goat anti-mouse, goat anti-rabbit or donkey anti-goat IgG for 2 h at room temperature. The membranes were subsequently washed with TBST, and the immunoreactive proteins were visualized using an enhanced chemiluminescent detection kit (CWBIO, China). The densitometric values of protein bands were quantified by using Image-Pro Plus 6.0.
P iκb
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom
P-IκB is a product offered by Cell Signaling Technology. It is a laboratory equipment used for scientific research purposes. The core function of P-IκB is to detect and measure the phosphorylation of IκB proteins.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (14 mentions)
P jnk, by Cell Signaling Technology (13 mentions)
P erk, by Cell Signaling Technology (12 mentions)
P nf κb p65, by Cell Signaling Technology (11 mentions)
β actin, by Cell Signaling Technology (10 mentions)
P nf κb, by Cell Signaling Technology (10 mentions)
Nf κb p65, by Cell Signaling Technology (9 mentions)
β actin, by Santa Cruz Biotechnology (9 mentions)
Nf κb, by Cell Signaling Technology (9 mentions)
P akt, by Cell Signaling Technology (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
P ikk, by Cell Signaling Technology (7 mentions)
Caspase 3, by Cell Signaling Technology (7 mentions)
Cox 2, by Cell Signaling Technology (6 mentions)
P erk1 2, by Cell Signaling Technology (6 mentions)
Cleaved caspase 3, by Cell Signaling Technology (6 mentions)
Cyclin d1, by Cell Signaling Technology (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Lipopolysaccharides (lps), by Merck Group (5 mentions)
P ikkα β, by Cell Signaling Technology (4 mentions)
83 protocols using p iκb
1
Jejunum Protein Extraction and Analysis
2
Oroxylin A Anti-inflammatory Signaling
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Oroxylin A was bought from Novartis Pharmaceuticals (Basel, Switzerland) and dissolved in dimethyl sulfoxide (DMSO) as a stock solution. Primary antibodies against CDK2, CyclinE, p27, p-p65, p65, and COX-2 were obtained from Cell Signaling (Beverly, MA, USA). Primary antibodies against E-cad, N-cad, Vimentin, p-IκB, IκB, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA). Secondary antibodies conjugated with horseradish peroxidase were obtained from Santa Cruz Biotechnology. All other reagents were purchased from Sigma-Aldrich (Louis, MO, USA).
3
Silencing RIPK4 in Bladder Cancer Cells
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Tumor cell line T24 (human bladder cells; American Type Culture Collection) was grown in Dulbecco’s modified Eagle’s medium (DMEM; CellGro) with 10% heat-inactivated fetal bovine serum (FBS) (CellGro) in 5% CO2 at 37°C. The oligoribonucleotide sequence of the siNC and human RIPK4 siRNA (siRIPK4) were as follows: 5′-GACACCAGCAAACUGAUGATT-3′ (sense) and 5′-UCAUCAGUUUGCUGGUGUCCC-3′ (antisense) (Shanghai GenePharma Co. Ltd., Shanghai, China). Fluorescein-tagged siRNA (FAM-siRNA) was synthesized using fluorescein modification of the 3′-end of the sense strand. Mouse antibodies recognizing RIPK4 and β-actin were obtained from Abcam (Cambridge, UK). Rabbit antibodies recognizing marker of proliferation Ki-67 (Ki-67), inhibitor of NF-κB (IκB) kinase subunit β (IKK), phospho (p)–IKK, IκB, p-IκB, and NF-κB p65 subunit (NF-κB-p65) antibodies were purchased from Cell Signaling Technology (Denver, MA, USA).
4
Magnolol Regulates Lipid Metabolism
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Magnolol, purity ≥98%, was purchased from Chengdu Reference Products (Chengdu, China). Dimethylsulfoxide (DMSO), oleic acid (OA), tyloxapol (Ty), and Oil Red O were obtained from Sigma-Aldrich (St. Louis, MO, USA). An Oil Red O stain kit (for cultured cells) was purchased from Solarbio Science & Technology (Beijing, China). Reactive oxygen species (ROS), TG, and total cholesterol (TC) assay kits were provided by Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). Human TNF-α enzyme-linked immunosorbent assay (ELISA) kits were provided by BioLegend (CA, USA). U0126, SB203580, SP600125, and LY290042 (specific inhibitors of ERK1/2, P38, JNK1/2, and AKT, respectively) and antibodies against AMPKα, P-AMPKα, AMPKβ, P-AMPKβ, adenosine ACC, P-ACC, P-ERK1/2, ERK1/2, P-JNK1/2, JNK1/2, P-P38, P38, IκB, P-IκB, and P-P65 were purchased from Cell Signaling Technology (Boston, MA, USA). AKT, P-AKT (Thr 308) and GAPDH antibodies were purchased from Affinity (OH, USA). Antibodies against P65, SREBP-1c and β-actin were purchased from Proteintech (Boston, MA, USA). PPARα and compound c (inhibitor of AMPK) were purchased from Abcam (Cambridge, MA, USA). HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies were provided by Boster (CA, USA). All other chemicals were of reagent grade.
5
Protein Expression Analysis in Retina and Cells
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The retina of mice and BV-2 or HRECs were collected and homogenized with lysis buffer, followed which centrifugation (1600 × g, 15 min) was performed at 4°C. Protein concentration was quantified in supernatant fluid by bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China). Separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to the PVDF membranes (GE Healthcare Europe GmbH, Freiburg, Germany). Proteins were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies (p-p65: ab76302; t-p65: ab32536; occludin: ab216327; claudin-1: ab180158; ZO-1: ab216880; VEGF: ab214424; CD31: ab182981; GAPDH: ab8245. Abcam, England. p-IκB: #2859, t-IκB: #9242, Iba-1: #17,198, Cell Signaling Technology, USA). Subsequently, HRP-conjugated secondary antibody (ab7090, abcam, England) was used to incubate the membranes for 1 h at 37°C. Protein bands were visualized by enhanced chemiluminescence reagent (Millipore Corp., Bedford, MA).
6
HSV-2 Strain 186 and Us2-Deficient Virus Propagation
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The wild-type HSV-2 strain 186 and the Us2-deficient (YY2) was obtained from the State Key Laboratory of Virology (Wuhan University) and both viruses were generated as described previously25 (link). The HSV-2 Us region expression plasmids were kind gifts of Dr. Yefu Wang, Wuhan University. NF-κB-luc plasmid were kind gifts of Dr. Qi Zhang, Wuhan University. Viruses were propagated on Vero cells and stored in aliquots at −80 °C until use. Viral titers were measured in Vero cells and expressed as plaque forming units (PFU)/ml and used for infection studies at a multiplicity of infection (MOI) of 1. UV-inactivated HSV-2 was obtained by exposure to UV irradiation for 20 min.
Antibody against β-tubulin and Lamin B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human and mice antibodies against TAK1, p-TAK1, IKKβ, p-IKKβ, IκB, and p-IκB were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against HSV-2 Us2 was purchased from WuXi AppTec (Shanghai, China). Antibody against Flag and HA were purchased from Sigma (St. Louis, MO, USA). All culture plasticware was obtained from Corning (Corning, NY, USA).
Antibody against β-tubulin and Lamin B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human and mice antibodies against TAK1, p-TAK1, IKKβ, p-IKKβ, IκB, and p-IκB were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against HSV-2 Us2 was purchased from WuXi AppTec (Shanghai, China). Antibody against Flag and HA were purchased from Sigma (St. Louis, MO, USA). All culture plasticware was obtained from Corning (Corning, NY, USA).
7
Immunofluorescence Imaging of Lung Signaling
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Tissues were processed as described above. Immunofluorescence studies were done according to a recently published study28 (link). In brief, lung sections were then incubated in blocking solution (5% bovine serum albumin (BSA) + 0.3% Triton X-100 in PBS) for 1 h, followed by incubation overnight at 4 °C with primary antibodies [pEGFR, pERK1/2, pAkt, pI-κB, (1:50 - 1:100 dilution); Cell Signaling, USA], diluted in 1% blocking solution. Subsequently, sections were washed and incubated with secondary antibody conjugated to Alexa Fluor 555 (Goat anti rabbit SFX kit; Life Technologies, USA, 1:400 dilution) for 2 h at room temperature in the dark. pI-κB was measured as a surrogate marker for NF-κB activation. After washes in PBS sections were stained with 4’,6 diamidino-2- phenylindole and mounted. Images were captured on a ZEISS LSM 700 confocal microscope and fluorescence intensity estimated in defined fields using Image J software package.
8
NF-κB Signaling Pathway Analysis
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Collected lung tissues were homogenized and lysed with RIPA buffer. Protein samples (50 μg) were separated by 10% SDS-PAGE and transferred onto a PVDF membrane, which was then blocked with 5% skimmed milk at room temperature for 1 h and incubated with primary antibodies against NF-κB (1:1000; #8242, Cell Signaling Technology, Danvers, MA, USA), P-NF-κB (1:1000; #3031, Cell Signaling Technology, Danvers, MA, USA), IκB (1:1000, #9242, Cell Signaling Technology, Danvers, MA, USA), P-IκB (1:1000, #2859, Cell Signaling Technology, USA), IL-33 (1:1000, #88513, Cell Signaling Technology, Danvers, MA, USA), anti-rabbit IgG (1:1000, #7074, Cell Signaling Technology, USA), and β-actin (1:1000, #4970, Cell Signaling Technology, USA). The signals were detected and visualized using an enhanced chemiluminescence (ECL) detection system. Bands were analyzed using Image J software (NIH, Bethesda, Rockville, MD, USA).
9
Antioxidant and Anti-inflammatory Mechanisms
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H2O2 (35 wt%) was purchased from Sigma−Aldrich (St. Louis, MO, USA). D−gal (≥99.0%) was obtained from Solarbio (Beijing, China). NMN (≥98.0%) and MET (≥98.0%) were obtained from Yuanye Biotechnology Co., Ltd. (Shanghai, China). According to a previously used method, SFE was isolated and purified [85 (link),86 (link)]. Primary antibodies against cleaved caspase−9, caspase−9, cleaved caspase−3, caspase−3, Cyclin D1, CDK6, Cyclin A, CDK2, Nrf2, HO−1, NQO−1, GCLM, TLR4, MyD88, p−NF−κB p65, p−IκB, and IL−6 were purchased from Cell Signaling Technology (Danvers, MA, USA). An antibody against β−actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibody HRP−conjugated goat anti−rabbit IgG and goat anti−mouse IgG were obtained from Abcam (Cambridge, MA, USA). Maintenance feed for mice (#SPF−F02−001) was purchased from SPF (Beijing) Biotechnology Co., Ltd.
10
RANKL Signaling in Cell Lines
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Cells were seeded at approximately 33% confluence in 6-well plates. The following day, they were washed and incubated in a medium without FBS. The next day, the medium was changed to 1.8 ml medium with or without 1 μM lapatinib followed by a 2-h incubation. Subsequently, 0.2 ml of medium with or without 300 ng/ml of RANKL (RANKL-LZ Amgen) was added to the wells. Ten minutes later, the extracts for immunoblots were prepared with modified RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate) containing 1× PhosSTOP and complete protease inhibitor cocktail (Roche), and protein concentrations determined with DC protein assay reagents (BIO-RAD). Fifteen micrograms of protein were resolved by SDS-PAGE and blotted into Immobilon-P 0.45 μm membranes (Millipore). Antibodies against the following proteins were used for probing: RANK (R&D Systems AF683), p-HER2 (#2249), HER2 (#2165), p-EGFR (#3777), EGFR (#4267), p-ERK1/2 (#9101), ERK1/2 (#9102), p-AKT (#4051), AKT (#9272), p-p65 (#3033), p65 (#8242), p-IκB (#9246), IκB (#9242) (from Cell Signaling), β-actin (sc-47778), and tubulin (Abcam ab21058).
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