Total protein was extracted with RIPA buffer (Beyotime, China). Then the concentrations in the samples were determined with BCA (Beyotime, China) methods. Next, the protein samples were separated with 10% SDS-PAGE gel (Beyotime, China). After that, the proteins were transferred to the PVDF membrane (Millipore, United States). Then, the membranes were blocked with a 5% skim milk powder solution (BD Bioscience, Regilait, France). After that, the membranes were incubated overnight with primary antibodies at 4°C. The primary antibodies used in this study were TNF-α (ABCAm, ab34674), IL-1β (ABCAm, ab200478), IL-18 (ABCAm, ab71495), NRF-2 (ABCAm, ab137550), HO-1 (ABCAm, ab137749) and GAPDH (ABCAm, ab181602). On Day 2, the membranes were washed with PBST three times. The membranes were then incubated with the second antibody for 2 hours. Finally, bands were developed with enhanced chemiluminescence reagents (Millipore, United States).
Ab137749
Manufactured by Abcam
Sourced in United Kingdom
Ab137749 is a laboratory equipment product. It is a core-shell magnetic nanoparticle designed for use in various bioanalytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Ab200478, by Abcam (1 mentions)
Ab71495, by Abcam (1 mentions)
Ab34674, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab137550, by Abcam (1 mentions)
Anti glial fibrillary acidic protein antibody z0334, by Agilent Technologies (1 mentions)
Anti arginase 1 bd610708 antibody, by BD (1 mentions)
Anti α tubulin t 5168 antibody, by Merck Group (1 mentions)
Ecl detection kit, by PerkinElmer (1 mentions)
Ab115210, by Abcam (1 mentions)
Ab196346, by Abcam (1 mentions)
Gtx100118, by GeneTex (1 mentions)
Gtx124246, by GeneTex (1 mentions)
Anti gapdh rabbit monoclonal antibody 14c10, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
7 protocols using ab137749
1
Western Blot Analysis of Inflammation and Antioxidant Markers
2
Antibody Sources and Applications
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Antibodies against IκBα (sc-371), JNK (sc-571), p65-NFκB (p65) (sc-732) and phospho-p38α MAPK (Thr 180/Tyr182) (sc-17852-R) were purchased from Santa Cruz Biotechnology (Palo Alto, CA, USA). Anti-phospho JNK (Thr183/Tyr185) (#4668) and anti-p38α MAPK (#9212) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-caspase-1 (p10) (ab 179515), iNOS (ab 15323), HO-1 (ab 137749), antibodies were purchased from Abcam (Cambridge, UK). Anti-Arginase-1 (BD610708) antibody was purchased from BD Bioscience (Madrid, Spain). Anti-Glial Fibrillary Acidic Protein (GFAP) antibody (Z0334) was obtained from DAKO (Glostrup, Denmark). Anti-NLRP3 (AG-20B-0006) was purchased from AdipoGen Life Science (Liestal, Switzerland) and anti-α-tubulin (T-5168) antibody was from Sigma-Aldrich (St Louis, MO, USA).
3
Western Blot Procedure for DENV Proteins
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The standard procedure of western blotting was performed as described previously [12 (link)]. The membranes were probed with monoclonal antibodies specific for DENV NS2B, (1:5000; GeneTex, GTX124246), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000; GeneTex, GTX100118), Nrf2 (1:3,000; GeneTex, GTX55732), HO-1 (1:3,000; Abcam, ab137749), Keap1 (1:1,000; Abcam, ab196346), and Bach1 (1:1,000; Abcam, ab115210). The ECL detection kit was used for signal detection (PerkinElmer, CT, USA).
4
Cinnamaldehyde Activates the NRF2 Pathway
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Cinnamaldehyde was obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) or in 100% ethanol (Wako, Osaka, Japan) at a concentration of 5 mM. Anti-NRF2 rabbit polyclonal antibody (H-300) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and horseradish peroxidase- (HRP-) linked anti-rabbit IgG antibody (#7074) was purchased from Cell Signaling Technology (Danvers, MA, USA). For Western blotting analysis, an anti-NRF2 rabbit polyclonal antibody (PA5-27882; Thermo Fisher Scientific), anti-HMOX1 rabbit polyclonal antibody (ab137749; Abcam), anti-NQO1 mouse monoclonal antibody (ab28947; Abcam), and anti-GAPDH (14c10) rabbit monoclonal antibody (#2118; Cell Signaling Technology) were used.
5
Western Blot Analysis of Nrf2 and Cardiac Markers
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Protein was isolated from cultured cells using lysis buffer that contained protease/phosphatase inhibitors. The quantification was determiend by the BCA Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Subsequently, after 5 min of denaturation at 95°C, samples in each group were separated by sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel. The PVDF membranes (EMD Millipore, Billerica, MA, USA) were used for the tank blot system. Next, the membrane was incubated with 5% skimmed milk containing 0.05% Tween-20 (TBST) at room temperature for 2 h. The primary antibodies were incubated at 4°C overnight: anti-Nrf2 (1:1,000, ab62352; Abcam, Cambridge, UK), anti-NQO-1 (1:800, ab34173), anti-HO-1 (1:1,000, ab137749), anti-BNP (1:500, ab19645), anti-β-MHC (1:500, ab23990), anti-GAPDH (ab9385, 1:5,000). The horseradish-peroxidase-coupled secondary IgG antibodies were incubated at room temperature for 1 h. ECL Western Blotting Substrate (Amersham; GE Healthcare, Chicago, IL, USA) was adopted to develop chemiluminescence signals using the ChemiDoc XRS Imaging System (Bio-Rad Laboratories, Inc.). Finally, the expression level was calculated by the densitometric analysis (Quantity One; Bio-Rad Laboratories, Inc.).
6
Western Blot Analysis of Betaine's Effects
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Western blot analysis was performed to analyze the influence of betaine treatment on the MAPK pathway, ROS-related enzymes and osteoclastogenesis-related markers. We used M-PER mammalian protein extraction reagent (Pierce, Rockford, IL, United States) to extract total protein from BMMCs. Twenty micrograms of protein sample per lane was loaded onto (11%) SDS-PAGE gels. When the protein sample reached the bottom of the gel, the power was cut off, electrophoresis was stopped, and the protein bands were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, 1620177). The primary antibodies included anti-P38 (Abcam, 1:1,000, ab170099), anti-P-P38 (Abcam, 1:1,000, ab195049), anti-ERK (Abcam, 1:1,000, ab17942), anti-P-ERK (Abcam, 1:1,000, ab201025), anti-JNK (Abcam, 1:1,000, ab179461), anti-P-JNK (Abcam, 1:1,000, ab124956), anti-TRAF6 (Abcam, 1:1,000, ab137452), anti-Nox1 (Abcam, 1:1,000, ab121009), anti-HO1 (Abcam, 1:1,000, ab137749), anti-catalase (Abcam, 1:1,000, ab217793), anti-TRAP (Abcam, 1:5,000, ab52750), anti-MMP9 (Abcam, 1:1,000, ab228402), and anti-Cathepsin K (Abcam, 1:1,000, ab187647), followed by the secondary antibody (Biosharp, 1:5,000, BL003A). The results were probed by chemiluminescence.
7
Intestinal Tissue Analysis of Apoptosis and Oxidative Stress
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Intestinal paraffin samples were cut into 4 μm sections, deparaffinized, and stained with primary antibodies for cleaved caspase-3 (#9661; Cell Signaling Technology), cleaved PARP (#94885; Cell Signaling Technology), LC3B (#3868; Cell Signaling Technology), p62 (ab56416; Abcam), 4-HNE (ab46545; Abcam), HO-1 (ab137749; Abcam), Trx1 (#2429; Cell Signaling Technology), Gpx4 (MABF1969; Millipore), γH2AX (#9718; Cell Signaling Technology), 53BP1 (ab172580; Abcam), p-NF-κB p65 (ab86299; Abcam), collagen I (sc-59772; Santa Cruz), collagen III (ab7778; Abcam), occludin (GTX85016; Genetex), ZO-1 (GTX108592; Genetex), and claudin-2 (ab53032; Abcam). Images of the stained sections were acquired using the Olympus BX51 microscope with an Olympus DP72 camera and cellSens Standard 1.14 software (Olympus, Germany).
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