After l -NAT treatment, the total protein was extracted from the BRL cells and the rat liver tissue using protein extraction buffer (RIPA Beyotime Biotechnology, Shanghai, China). The cells were washed twice with ice-cold PBS (pH 7.4) and RIPA buffer (Solarbio, Beijing, China). Proteins were subjected to SDS-PAGE with a 10% running gel and then transferred to a PVDF membrane. The samples were incubated with the following antibodies: anti-NF-κB (1:500, Bioss, Beijing, bs-0465R); anti-TLR-4 (1:500, Bioss, Beijing, bs-1021R); anti-RIP2 (1:500, Santa Cruz); and anti-GAPDH (1:2000, Proteintech, 10494-1-AP), respectively. The details for the analysis have been described previously (Huang et al. 2015 (link)). Western blot images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). GAPDH was used as the internal reference protein.
Anti nf κb
Manufactured by Bioss Antibodies
Sourced in China
Anti-NF-κB is a lab equipment product used for the detection and measurement of the NF-κB (Nuclear Factor Kappa B) protein. NF-κB is a transcription factor that plays a critical role in regulating the immune response, inflammation, and cell survival. The Anti-NF-κB product can be used to study the expression and activity of NF-κB in various experimental models and biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti tlr4, by Bioss Antibodies (3 mentions)
Anti asc antibody, by Bioss Antibodies (2 mentions)
Anti nlrp3 antibody, by Bioss Antibodies (2 mentions)
Anti rip2, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Anti myd88, by Bioss Antibodies (1 mentions)
Bradford protein assay kit, by Keygen Biotech (1 mentions)
Enhanced chemiluminescence detection kit, by Beyotime (1 mentions)
Anti phospho nf κb, by Santa Cruz Biotechnology (1 mentions)
Pvdf transfer membrane, by Merck Group (1 mentions)
Bradford easy protein quantitative kit, by Transgene (1 mentions)
Anti gapdh, by Transgene (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
Anti tsp 1, by Thermo Fisher Scientific (1 mentions)
Anti phospho and total p38, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti total akt, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti pcna, by Abcam (1 mentions)
8 protocols using anti nf κb
1
Quantitative Analysis of NF-κB, TLR-4, and RIP2 in L-NAT Treated Cells
2
Macrophage Protein Extraction and Western Blot
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Macrophages were collected and proteins were extracted according to the manufacturer’s handbook (KeyGEN BioTECH, Nanjing, China). Protein concentrations in each sample were determined by Bradford Protein Assay Kit (KeyGEN BioTECH, Nanjing, China). The proteins were then separated with 10 % SDS-PAGE and transferred on nitrocellulose membrane, which was blocked overnight with 5 % (w/v) non-fat dry milk in TBST (20 mmol/l Tris-HCl (pH 7.6), 150 mmol/l NaCl and 0.02 % Tween 20). Membranes were then washed three times for 10 min in TBST and incubated for 1 hr at 37 °C with rabbit polyclonal anti-MyD88, anti-NF-κB and anti-β-actin primary antibodies (Bioss, Beijing, China). In addition, anti-β-actin was used to detect β-actin expression as a quantitative control. After being washed three times for 10 min in TBST the membranes were incubated for 1 hr at RT with horseradish peroxidase (HRP)-conjugated secondary antibody. The membranes were then washed again three times in TBST and protein bands were visualized with the ECL enhanced chemiluminescent (Hai-Gene, Harbin).
3
Western Blot Analysis of MAC-T Cells
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Protein lysates from the MAC-T cells were prepared with ice-cold PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea) in accordance with the manufacturer's instructions. Lysates were precipitated at 1200 rpm for 10 min at 4°C. The total protein content was determined using the Bradford Easy Protein Quantitative Kit (TransGene). Equal amounts of protein extracts in lysis buffer were separated using 4%–12% polyacrylamide gels (German, Sigma-Aldrich) and then transferred onto a nitrocellulose membrane. Resolved proteins were blotted onto PVDF transfer membranes (Millipore Co., Bedford, MA) and then blocked with 10% nonfat milk in TBST. The membranes were incubated with anti-CYP1A1 (Sangon Biotech, Shanghai, China), anti-NF-κB (Bioss, Beijing, China), anti-phospho-NF-κB (Santa Cruz Biotechnology lnc., Santa Cruz, CA, USA), and anti-GAPDH (TransGene) antibodies at 4°C overnight. The membranes were washed three times with TBST for 5 min before incubation with HRP-conjugated secondary antibodies. Finally, the immunoreactive proteins were visualized using an enhanced chemiluminescence detection kit (Beyotime).
4
Western Blot Analysis of Protein Targets
5
VIP-Induced Osteoclastogenesis Regulation
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VIP was purchased from Nanjing Peptide Biotechnology Inc. (Nanjing, China). Fetal bovine serum (FBS), a modification of Minimum Essential Medium (a-MEM), and L-glutamine were supplied by Invitrogen (Carlsbad, USA). Oligonucleotide primers were ordered from Sangon Biotech (Shanghai, China). TaqMan Universal PCR Master Mix and probes were obtained from Applied Biosystems (Foster City, CA). anti-RANKL, anti-RANK, anti-OPG, anti-NF-κB, anti-interleukin 6 (IL-6), anti-ERK, anti-carbonic anhydrase II (CAII), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primary, and anti-IgG-horseradish peroxidase secondary antibodies were purchased from Bioss Biotechnology, Inc. (Beijing, China).
6
Protein Expression Analysis in Colon Tissues
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Cells were lysed by NP-40 Lysate (Beyotime Institute of Biotechnology, Haimen, China), and the total protein from the colon tissues of each group was extracted using radio-immunoprecipitation assay (RIPA) (Beyotime) lysis buffer, and protein concentrations were determined using the bicinchoninic acid (BCA) (Beyotime) method. Forty micrograms of total protein from each sample was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis electrophoresis, followed by transfer to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was incubated with primary antibodies anti-IL-1β, anti-NF-κB, anti-cleaved caspase-3, anti-Bax, and anti-Bcl-2 (1:1000 dilution, bioss, Beijing, China) overnight at 4°C. Subsequently, the membrane was incubated with goat anti-rabbit IgG-HRP (1:5000, Beyotime) at room temperature for 1 h, followed by chromogenic detection using the enhanced chemiluminescence method. The film was scanned and analyzed by Gel-Pro-Analyzer software to determine the optical density value of the target bands using β-actin as the internal control.
7
Immunofluorescence Analysis of Inflammasome Pathway
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BRL cells were exposed to H2O2 with or without L-NAT, fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100-PBS for 15 min. Blocking was done in 5% goat serum for 30 min at 37 °C to inactivate endogenous peroxidase. Subsequent, the cells were incubated with anti-ASC antibody (1:200; Bioss), anti-NLRP3 antibody (1:200; Bioss), anti-IL-1β antibody (1:200; Bioss), anti-Caspase-1 antibody (1:100; Santa), anti-TLR4 (1:500; Bioss), and anti-NF-κB (1:500; Bioss) at 4 °C overnight and then incubated with FITC-conjugated secondary antibodies (1:150) at 37 °C for 30 min. DAPI staining was performed and image were taken using confocal microscopy (Olympus FV500; Olympus, Tokyo, Japan). Liver tissue from the sham group, I/R group and I/R + L-NAT group were made frozen sections of 10 µm thickness. And then immunofluorescence staining was performed using the method described above.
8
Quantitative Protein Analysis in Liver
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For protein extraction, each of 100 mg liver tissues were homogenized on ice in 1 ml RIPA lysis buffer with protease inhibitor mix PMSF. Lysates were obtained by centrifugation at 15,000 rpm for 15 min at 4 °C. The protein concentration was quantified by BCA method. Subsequently, protein samples were subjected to 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane, and then incubated with the following antibodies: anti-ASC antibody (1:200; Bioss), anti-NLRP3 antibody (1:200; Bioss), anti-TLR4 (1:500; Bioss) and anti-NF-κB (1:500; Bioss). Finally, the bands were observed using chemiluminescence (ECL) system according to the manufacturer’s instructions. Western blotting bands were analyzed using optical density scanning and Image Lab software (Bio-Rad). GAPDH (1:1000; Proteintech) was used as the loading control.
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