RAW264.7 cells were treated with dead or live E. tarda as above for 0 h, 1 h, 2 h, and 4 h. The cells were lysed on ice for 30 min with RIPA lysis buffer (Beyotime, Beijing, China) containing phosphatase inhibitor cocktail (Beyotime, Beijing, China). The cell lysates were mixed with SDS-PAGE loading buffer and boiled at 100 °C for 10 min. The samples were then subjected to SDS-PAGE, and the separated proteins were electro-transferred from gels to nitrocellulose blotting membranes (GE healthcare, Germany). The membranes were soaked in TBST (20 mM Tris, pH 7.5; 500 mM NaCl; 0.1% Tween 20) containing 5% bovine serum albumin (Solarbio, Beijing, China) for 2 h. The membranes were incubated with rabbit anti-phospho-NF-κB p65 (Ser536) monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-NF-κB p65 monoclonal antibody (ABclonal, Wuhan, China), or anti-β-actin monoclonal antibody (ABclonal, Wuhan, China) at 4 °C for overnight. After extensive washing with TBST, the membranes were incubated with HRP-conjugated anti-rabbit antibody (Abcam, Cambridge, UK) for 1h at room temperature. The membranes were washed with TBST for three times and incubated with enhanced chemiluminescence (ECL) solution (Beyotime, Beijing, China). The membranes were visualized using a GelDoc XR System (Bio-Rad, Hercules, CA, USA).
Rabbit monoclonal anti phospho nf κb p65 ser536 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Rabbit monoclonal anti-phospho NF-κB p65 (ser536) antibody is a laboratory reagent used to detect the phosphorylation of the NF-κB p65 subunit at serine 536. This antibody is designed for use in Western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase and phosphatase inhibitor, by Roche (2 mentions)
Ecl prime western blotting detection system, by GE Healthcare (2 mentions)
Ecl plus blocking agent, by GE Healthcare (2 mentions)
Anti igg antibody conjugated with horseradish peroxidase, by GE Healthcare (2 mentions)
Rabbit monoclonal anti p65 antibody, by Cell Signaling Technology (2 mentions)
Mouse monoclonal anti α tubulin antibody, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose blotting membrane, by GE Healthcare (1 mentions)
Phosphatase inhibitor cocktail, by Beyotime (1 mentions)
Hrp conjugated anti rabbit antibody, by Abcam (1 mentions)
Ecl solution, by Beyotime (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Rabbit anti phospho stat1 tyr 701 monoclonal antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti gapdh polyclonal antibody, by Proteintech (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
Hybond p, by GE Healthcare (1 mentions)
5 protocols using rabbit monoclonal anti phospho nf κb p65 ser536 antibody
1
Inflammatory Pathway Activation by E. tarda
2
Phosphorylation Analysis of Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were treated with FIN and cells were harvested at different time points and lysed with lysis buffer for the detection of phosphorylation of transcription factors, phosphatase inhibitor was added into the lysis buffer. The lysate were then subjected to electrophoresis, followed by transferring onto the membrane and detection with the primary antibodies including rabbit anti-STAT1 polyclonal antibody (abcam, The United Kingdom), rabbit anti-STAT6 polyclonal antibody (ABclonal, Baltimore, MD), rabbit anti-phospho STAT1 (Tyr 701) monoclonal antibody (Cell Signaling Technology, Danvers, MA), rabbit anti-phospho STAT6 (Tyr 641) polyclonal antibody (ABclonal), rabbit anti-phospho NF-κB p65 (Ser536) monoclonal antibody (Cell Signaling Technology), rabbit anti-COX2 polyclonal antibody (R&D systems), rabbit anti-nuclear matrix protein p84 monoclonal antibody (abcam), rabbit anti-β-Actin polyclonal antibody (Proteintech) and rabbit anti-GAPDH polyclonal antibody (Proteintech).
3
Western Blot Analysis of Macrophage Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In Western Blot analysis, whole cell lysate was prepared from primary culture of macrophages treated with indicated stimuli in the Results or Figure Legends by RIPA buffer (Sigma Aldrich, St. Louis, MO) supplemented with proteinase and phosphatase inhibitors (Roche, Indianapolis, IN). After Sodium Dodecyl Sulfate- Polyacrylamide gel Electrophoresis (SDS-PAGE), separated proteins were transferred to a PVDF membrane (Immobilon-P, Merck) and blocked with an ECL plus blocking agent (GE healthcare, Buckinghamshire, UK). Blotted membranes were, then, incubated with primary antibodies followed by incubation with anti-IgG antibody conjugated with horseradish peroxidase (GE healthcare). Finally, the signal was detected by the chemiluminescent reagent (ECL Prime Western Blotting Detection System, GE healthcare). α-Tubulin was served as an internal control.
Primary antibodies used in this study are as follows: rabbit monoclonal anti-p65 antibody (Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-phospho NF-κB p65 (ser536) antibody (Cell Signaling Technology), mouse monoclonal anti-IκBα antibody (Cell Signaling Technology), mouse monoclonal anti-α-tubulin antibody (Sigma Aldrich).
Primary antibodies used in this study are as follows: rabbit monoclonal anti-p65 antibody (Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-phospho NF-κB p65 (ser536) antibody (Cell Signaling Technology), mouse monoclonal anti-IκBα antibody (Cell Signaling Technology), mouse monoclonal anti-α-tubulin antibody (Sigma Aldrich).
4
Antibody Verification for Signal Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); rabbit anti-phospho-ERK1/2 MAPK (Thr202/Tyr204) antibody (#9101), rabbit anti-ERK1/2 MAPK antibody (#9102), rabbit monoclonal anti-phospho-NF-κB p65 (Ser536) antibody (#3033), rabbit monoclonal anti-NF-κB p65 antibody (#8242), rabbit anti-phospho-IκBα (S32) antibody (#2859), rabbit IκBα (44D4) antibody (#44D4), rabbit monoclonal anti-cyclooxygenase (COX) 2 (D5H5) XP antibody (#12282), and rabbit monoclonal histone H3 antibody (D1H2) XP (#4499). In addition, rabbit anti-phospho p38 MAPK antibody (Thr180/Tyr182) was obtained from Promega Corporation (Madison, WI, USA) and mouse monoclonal anti-p38 MAPK (p38/SAPK2) antibody (#612168) was obtained from BD Biosciences (San Jose, CA, USA). Mouse anti-α tubulin (p38/SAPK2) antibody (#612168) was obtained from Thermo Fisher Scientific (Waltham, MA, USA), Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Merck Millipore (Burlington, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (AP132P) and HRP-conjugated goat anti-mouse IgG/IgM antibody (AP308P) were obtained from Chemicon International (Temecula, CA, USA).
5
Western Blot Analysis of NF-κB Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysate was prepared by a RIPA buffer (Sigma Aldrich) supplemented with proteinase and phosphatase inhibitors (Roche, Indianapolis, IN). Protein concentration was, then, determined by a bicinchoninic acid (BCA) method (Pierce BCA Protein Assay Kit, Thermo Scientific, Waltham, MA). After sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE), separated proteins were transferred to a PDVF membrane (Hybond-P, GE healthcare, Buckinghamshire, UK) and blocked with an ECL plus blocking agent (GE healthcare). The membranes were incubated with primary antibodies followed by incubation with an anti-IgG antibody conjugated with horseradish peroxidase (GE healthcare). Finally, the signal was detected by a chemiluminescent reagent (ECL Prime Western Blotting Detection System, GE healthcare). α-Tubulin was served as an internal control.
Antibodies used in Western blot analysis are as follows: rabbit monoclonal anti-p65 antibody (Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-phospho NF-κB p65 (Ser536) antibody (Cell Signaling Technology), and mouse monoclonal anti-α-tubulin antibody (Sigma Aldrich).
Antibodies used in Western blot analysis are as follows: rabbit monoclonal anti-p65 antibody (Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-phospho NF-κB p65 (Ser536) antibody (Cell Signaling Technology), and mouse monoclonal anti-α-tubulin antibody (Sigma Aldrich).
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!