LOB2 and LOB3 were provided as an in-kind contribution from Prairie Tide Diversified Inc. (PTD, Saskatoon, SK Canada). The human THP-1 monocytic cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). Roswell Park Memorial Institute (RPMI)-1640 supplemented with l -glutamine and HEPES, fetal bovine serum (FBS), and penicillin–streptomycin (P/S, 10 000 IU mL−1) were purchased from Gibco BRL (Life Technologies Ltd., Paisley, UK). Lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), phosphate buffered saline solution (PBS), 2-mercaptoethanol, phorbol 12-myristate 13-acetate (PMA), and other chemicals were of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA). Protease and phosphatase inhibitor cocktail, radio immunoprecipitation assay (RIPA) lysis buffer, BCA protein assay kit, Griess reagent kit and instant enzyme-linked immunosorbent assay (ELISA) kits of human IL-1β, IL-6 and TNF-α were from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies against β-actin, cyclooxygenase-2 (COX-2), p-IKKα/β, p-IκBα and p-p65-NF-κB were obtained from Cell Signaling Technology (Danvers, MA, USA).
P nf κb p65
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom
P-NF-κB p65 is an antibody that detects the phosphorylated form of the NF-κB p65 subunit. NF-κB is a transcription factor that plays a crucial role in regulating the immune response, cell survival, and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Nf κb p65, by Cell Signaling Technology (109 mentions)
P iκbα, by Cell Signaling Technology (58 mentions)
β actin, by Cell Signaling Technology (38 mentions)
P jnk, by Cell Signaling Technology (37 mentions)
P akt, by Cell Signaling Technology (35 mentions)
P erk, by Cell Signaling Technology (27 mentions)
P erk1 2, by Cell Signaling Technology (27 mentions)
Gapdh, by Cell Signaling Technology (26 mentions)
Erk1 2, by Cell Signaling Technology (23 mentions)
P ikkα β, by Cell Signaling Technology (20 mentions)
P p38 mapk, by Cell Signaling Technology (15 mentions)
P stat3, by Cell Signaling Technology (15 mentions)
Cleaved caspase 3, by Cell Signaling Technology (14 mentions)
Lipopolysaccharides (lps), by Merck Group (12 mentions)
P38 mapk, by Cell Signaling Technology (12 mentions)
P iκb, by Cell Signaling Technology (11 mentions)
Cox 2, by Cell Signaling Technology (10 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
β actin, by Santa Cruz Biotechnology (10 mentions)
Pvdf membrane, by Merck Group (10 mentions)
235 protocols using p nf κb p65
1
Inflammatory Response Modulation in THP-1 Cells
2
Immunoblot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We conducted immunoblot analyses of HK-2 cell lysates as described previously [8 (link)]. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellu-lose membranes, and incubated with antibodies against the following proteins: VDR (Santa Cruz Biotechnology); COX-2, Akt, pS473 Akt, pp65 NF-κB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, MA, USA); EP4 and p65 NF-κB (Abcam, Cambridge, UK). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) or anti-mouse IgG (Invitrogen Biotechnology) and signal densities were detected and quantified using the ChemiDoc XRS Image system (Bio-Rad Laboratories).
3
Immunoblot Analysis of Kidney and Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed immunoblot analyses for mice kidneys and HK-2 cell lysates as described previously [12 (link)]. The proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with antibodies against the following proteins: VDR (Santa Cruz Biotechnology); COX-2, Akt, pS473 Akt, CREB, pS133 CREB, pp65 NF-κB, BCL-2-associated X (Bax), B-cell leukemia/lymphoma 2 (Bcl-2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, MA, USA); EP4, p65 NF-κB, superoxide dismutase (SOD) 1 and SOD2 (Abcam); β-actin (Sigma-Aldrich). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Invitrogen Biotechnology, CA, USA) and positive bands were detected and analyzed using the ChemiDoc XRS Image system (Bio-Rad Laboratory).
4
Protein Expression Analysis via Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were collected in a RIPA buffer (R0010, Solarbio, China) and separated by 12% SDS‒PAGE (Bio-Rad, China). After being transferred to PVDF membranes, blocked with 5% milk, the membranes were incubated with appropriate primary antibodies overnight at 4 °C. The primary antibodies included CTSK (1:800, #DF6614, Affinity, China), CD68 (1:1000, #14-0688-82, ThermoFisher), CD163 (1:1000, #14-1639-82, ThermoFisher), CD200R (1:1000, #DF4715, Affinity), p-p65 NF-κB (1:1000, #3039, Cell Signaling Technology), p65 NF-κB (1:1000, #3034, Cell Signaling Technology), and GAPDH rabbit antibody (1:1000, #5174, Cell Signaling Technology). After washing with TBS-0.01% Tween 20, the membranes were incubated with secondary antibody (1:1000, #14708, Cell Signaling Technology) for 2 h at 25 °C. After washing, the blots were visualized using an enhanced chemiluminescence reagent (D085075, Bio-Rad, China).
5
Western Blot Analysis of GR, NF-κB, and MAPK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured HNECs were seeded in a six-well plate, lysed, and then the protein was extracted by the RIPA lysis buffer combined with the protease and phosphatase inhibitor mix usage per well and quantified by the BCA kit usage. Proteins were separated using 10% polyacrylamide gel electrophoresis (PAGE). Every well was loaded with the protein sample and a protein marker on both sides, 5–10 µL, and 5 µL, respectively. After that, electrophoresis was done for 110 minutes at a 90 V constant voltage. The membrane was incubated with blocking buffer and incubated with GRα antibody ((4 μg.mL-1; Invitrogen, PA1516, USA), GRβ antibody (1:500; Invitrogen, PA3-514), p-P65 NF-κB (1:1000; Cell Signaling Technology, 3033T), p-P38 MAPK (1:1000; Cell Signaling Technology, 4511),
and β-actin rabbit monoclonal antibody (1:3000; Beyotime, AF5003, China) overnight at 4 °C. The membranes were treated with the HRP-labeled secondary goat anti-rabbit IgG antibody.
Immunoreactive proteins were measured using BeyoECL plus an Enhanced Chemiluminescence Western blot detection system.
and β-actin rabbit monoclonal antibody (1:3000; Beyotime, AF5003, China) overnight at 4 °C. The membranes were treated with the HRP-labeled secondary goat anti-rabbit IgG antibody.
Immunoreactive proteins were measured using BeyoECL plus an Enhanced Chemiluminescence Western blot detection system.
6
Splenic B Cell R848 Stimulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After stimulation of splenic bee cells with 1 mg/ml of R848 for indicated times, cells were washed with cold PBS and lysed with cold lysis buffer (Cell signaling Technology) supplemented with Halt’s protease and phosphatase inhibitors (Thermo Fisher Scientific). Total protein was determined using Pierce BCA protein assay kit. pIRAK4 and pIRAK1 antibodies were generated at Genentech [52 (link)], and all other antibodies, including p-IκBα, p-p65 NFκB, p-p38 and p-JNK, actin, and anti-rabbit IgG–HRP linked antibody were purchased from Cell Signaling Technology. Detection was done using SignalFire™ ECL chemiluminescence substrate from Cell Signaling Technology.
7
TAK1 and TAZ Regulation in Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TAK1 inhibitor takinib (#1111556-37-6) was purchased from Sigma-Aldrich. TAZ overexpression lentiviral particles (TAZ-Flag: pLenti-EF1A-WWTR1-CMV-Flag-Puro) and TAK1 overexpression lentiviral particles (TAK1-Flag: pLenti-EF1A-TAK1-CMV-Flag-Puro) were generated by PPL (GeneBio Technology Inc. Nanjing, China). TAZ silencing lentivirus (shTAZ: pPLK/GFP+Puro-TAZ) was purchased from Shanghai Genechem Co. Ltd. (Shanghai, China). Silencing of shTAZ was performed with the following sequences: (1) 5ʹ-CCGGGCGATGAATCAGCCTCTGAATCTCGAGATTCAGAGGCTGATTCATCGCTTTTTG-3ʹ; (2) 5ʹ-CCGGGCGTTCTTGTGACAGATTATACTCGAGTATAATCTGTCACAAGAACGCTTTTTG-3ʹ; and (3) 5ʹ-CCGGCCAGGAACAAACGTTGACTTACTCGAGTAAGTCAACGTTTGTTCCTGGTTTTTG-3ʹ. Specific antibodies against β-actin (AC-15) (sc-69879) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against p-TAZ (#59971), TAK1 (#D94D7), p-TAK1 (#9339), I-κBα (#5209), p65 NF-κB (#8242) and p-p65 NF-κB (#3033) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against TAZ (23306-1-AP) and Flag (66008-2-Ig) were purchased from Proteintech (Rosemont, IL, USA), and NFATc1 (#6677), antibodies against p-p65 NF-κB (#5088) were purchased from Bioworld Technology Inc. (St. Louis Park, MN, USA), and antibodies against TAZ (#242313) was purchased from Abcam (Cambridge, UK).
8
Quantitative RT-PCR and Immunoblot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oligonucleotide primers were synthesized by Integrated DNA Technologies and are listed in Table 1 . RNA was subjected to conventional and quantitative RT-PCR analyses using selective primer sequences as we described previously [25 (link)]. The universal ERβ PCR amplimer set amplifies wild-type and all subtypes. Protein extracts were subjected to immunoblot analysis with antibodies to p-Src, c-Src (32G6), p-IGF-ΙR, IGF-ΙR, cyclin D1, p-p65 NF-κB, NF-κB, IκB, and p-IκB (Cell Signaling Technology); PSA, ETV5, or GAPDH (Santa Cruz Biotechnology); ERβ; or TMPRSS2 (Abcam, Cambridge, MA, USA). Immune complexes were detected with appropriate secondary antibodies and chemiluminescence reagents (Pierce Biotechnology, Rockford, IL, USA). Densitometry of immunoblot signals was quantified using the VersaDoc™ imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
9
Quantitative Immunoblotting Analysis
10
Protein Extraction and Western Blot Quantification
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!