Lung tissues were homogenized by a homogenizer and lysed using RIPA buffer containing a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The protein concentration of each group was determined using BCA kits. Protein samples (30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and subsequently transferred onto PVDF membranes, which were blocked by 5% skim milk for 1 hour at room temperature. After rinsing three times, the blots were incubated overnight at 4°C with the following primary antibodies: anti-TLR4 (1:1,000; Cell Signaling Technology), anti-NF-κB p65 (1:1,000; Cell Signaling Technology), anti-phospho-IκBα (1:1,000; Cell Signaling Technology), anti-IκBα (1:1,000; Santa Cruz), and anti-Cit-H3 (1:1000, Abcam). The membranes were washed three times, incubated with horseradish peroxidase–conjugated secondary antibodies (1:3,000; Sigma-Aldrich), and then visualized by an enhanced chemiluminescence kit (ECL plus). We used anti-GAPDH (1:2,000; Cell Signaling Technology), anti-β-actin (1:10,000; Sigma-Aldrich) and anti-Histone-H3 (1:1500; Sigma) as internal controls. To measure the relative ratio of protein expression, band intensities were quantified by Image-Lab software (Bio-Rad).
Anti cith3
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-CitH3 is a laboratory product that detects citrullinated histone H3 (CitH3). CitH3 is a post-translational modification involved in various cellular processes. This product can be used to measure CitH3 levels in samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Anti gapdh, by Proteintech (4 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti pad4, by Abcam (2 mentions)
Anti traf6, by Santa Cruz Biotechnology (2 mentions)
Ecl system, by Cytiva (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Biospectrum imaging system, by Analytik Jena (2 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti tlr4, by Cell Signaling Technology (1 mentions)
Anti histone h3, by Merck Group (1 mentions)
Anti phospho iκbα, by Cell Signaling Technology (1 mentions)
Anti iκbα, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
16 protocols using anti cith3
1
Quantifying Lung Protein Biomarkers
2
Western Blot Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates or mouse tissue homogenates were separated by electrophoresis on 10% SDS-PAGE, transferred on PVDF membranes (Merck Millipore, Burlington, MA, USA), blocked in 5% of non-fat dry milk, and incubated with primary antibodies including anti-Bax (1:2000, Abcam), anti-Beclin-1 (1:1000, Cell Signaling), anti-CitH3 (1:1000, Abcam), anti-LC3 (1:1000, Cell Signaling), anti-mTOR (1:1000, Cell Signaling), anti-p53 (1:1000, Santa Cruz), anti-PAD4 (1:1000, Abcam), anti-TLR4 (1:000, Santa Cruz), anti-TRAF6 (1:1000, Santa Cruz), anti-GAPDH (1:5000, Proteintech, Rosemont, IL, USA) or anti-β-actin antibodies (1:5000, Sigma-Aldrich) at 4 °C for 16 h. After washing, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000, Jackson Immunoresearch, West Grove, PA, USA) at RT for 2 h. Signal expression of protein-antibody complexes was detected by ECL system (Amersham Pharmacia Biotech, Buckinghamshire, UK) and visualized with Biospectrum imaging system (UVP, Upland, CA, USA). Relative protein expression levels were measured by Image J (National Institute of Health, Bethesda, MD, USA).
3
Neutrophil NET Formation Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated human neutrophils were plated and stimulated on Nunc LabTek chambers (Thermo Fisher Scientific, Waltham, MA, USA) with LPS (10 µg/ml) for 3 h. After stimulation, the cells were then washed and fixed with paraformaldehyde (4%) for 20 min and blocked in 1% bovine serum albumin for 1 h before being incubated with anti-Cit-H3 (Abcam) and anti-TREM-1 (Bioss), while DNA was stained with TO-PRO-3 at 4 °C overnight. After being washed with PBS, the coverslips were mounted with Vectashield (Vector Laboratories, CA, USA) and visualized through sequential scanning on an SP5 confocal microscope (Leica, Wetzlar, Germany). Images were processed using LAS-AF Lite software (Leica, Wetzlar, Germany).
4
Western Blot Analysis of Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from lysis buffer containing a protease inhibitor, and the level of protein was quantified with a protein quantification kit (TransGen Biotech, Beijing, China). Then, equivalent protein samples were separated by SDS-PAGE gels and transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany), which were blocked in Protein-free fast blocking solution (Boster, China) and cultivated with primary antibody for 12h at 4 ℃. After washing three times with wash buffer, the membrane was incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 37C°. Protein bands were intuitively visualized by an enhanced chemiluminescence (ECL) imaging system (Bio-Rad Laboratories, CA, USA) and quantified with ImageJ software (Rawak Software Inc., Germany). Principal primary antibodies were as follows: rabbit polyclonal anti-CitH3 (1:1000, Abcam, UK), rabbit polyclonal anti-NLRP3 (1:1000, Abcam, UK), rabbit polyclonal anti-GSDMD (1;1000, Cell single Technology, USA), rabbit polyclonal anti-caspase-1 (1:1000, Abcam, UK), rabbit polyclonal anti-H3 (1:1000 dilution, Immunoway Biotechnology, USA), rabbit polyclonal anti-caspase-1 p20 (1:500, Novus Biologicals LLC, USA), and rabbit polyclonal anti-GAPDH (1:1000, Proteintech, China).
5
Protein Isolation and Western Blot Analysis
6
Immunoblotting Analysis of Mouse Lung Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse lung tissue homogenates were separated by electrophoresis on 10 to 15% SDS-PAGE, transferred on PVDF membranes (Merck Millipore, Burlington, MA, USA), blocked in 5% of non-fat dry milk, and incubated with primary antibodies including anti-CitH3 (1:1000, citrulline R2 + R8 + R17, Abcam, Cambridge, UK), anti-TRAF6 (1:1000, Santa Cruz), anti-IRAK-1 (1:1000, Santa Cruz), anti-PAD4 (1:1000, Abcam), anti-HMGB1 (1:1000, Abcam) or anti-β-actin antibodies (1:5000, Sigma-Aldrich) at 4 °C for 18 h. After washing, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000, Jackson Immunoresearch, West Grove, PA, USA) at RT for 2 h. Signal expression of protein-antibody complexes was detected by ECL system (Amersham Pharmacia Biotech, Buckinghamshire, UK) and visualized with Biospectrum imaging system (UVP, Upland, CA, USA). Relative protein expression levels were measured by Image J (National Institute of Health, Bethesda, MD, USA).
7
Investigating Signaling Pathways in EGFR-Mediated Epithelial-Mesenchymal Transition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies used were anti‐phospho‐EGFR1068 (Cell Signaling Technology, #3777), anti‐EGFR (Cell Signaling Technology, #4267), anti‐ERK (Cell Signaling Technology, #4695), anti‐phospho‐ERK (Cell Signaling Technology, # 4370), anti‐Snail (Cell Signaling Technology, #3879), anti‐E‐cadherin (Cell Signaling Technology, #14472), anti‐vimentin (Cell Signaling Technology, # 5741), anti‐N‐cadherin (Cell Signaling Technology, #13116), anti‐IL‐1β (Cell Signaling Technology, #12703), anti‐Cit‐H3 (Abcam, ab5103), anti‐H3 antibodies (Abcam, ab1791) and anti‐β‐actin antibody (Abcam, ab8226). The inhibitors erlotinib, SHC772984 and Sivelestat (ONO‐5046) were purchased from Selleck Chemicals (Selleck Chemicals, Houston, TX, USA).
8
Western Blot Analysis of Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates and supernatants were routinely processed as described previously. Additionally, cell lysates were prepared according to a previous detailed description39 (link) for ASC oligomerization.
Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore; MA, USA) after electrophoresis. The membranes were blocked with 8% skimmed milk for 1 h at RT and incubated with primary antibodies, including anti-cit-H3 (1:1000, Abcam, MA, USA), anti-NE (1:1000, R&D Systems, MN, USA), anti-cleaved caspase-1 (1:1000, GeneTex; CA, USA), anti-IL-1β (1:1000, GeneTex; CA, USA), anti-ASC (1:1000, GeneTex; CA, USA) and anti-GAPDH (1:1000, Proteintech, Wuhan, China) antibodies, overnight at 4°C. After three washes (Tris-buffered saline plus 0.05% Tween-20), the membranes were incubated with a horseradish peroxidase-conjugated goat-anti rabbit or mouse secondary antibody (SAB; MD, USA) for 1 h at RT on a shaker. Signals were detected with ClarityMax Western ECL Substrate (Bio-Rad, CA, USA) and quantified by ImageJ software (Rawak Software Inc., Germany).
Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore; MA, USA) after electrophoresis. The membranes were blocked with 8% skimmed milk for 1 h at RT and incubated with primary antibodies, including anti-cit-H3 (1:1000, Abcam, MA, USA), anti-NE (1:1000, R&D Systems, MN, USA), anti-cleaved caspase-1 (1:1000, GeneTex; CA, USA), anti-IL-1β (1:1000, GeneTex; CA, USA), anti-ASC (1:1000, GeneTex; CA, USA) and anti-GAPDH (1:1000, Proteintech, Wuhan, China) antibodies, overnight at 4°C. After three washes (Tris-buffered saline plus 0.05% Tween-20), the membranes were incubated with a horseradish peroxidase-conjugated goat-anti rabbit or mouse secondary antibody (SAB; MD, USA) for 1 h at RT on a shaker. Signals were detected with ClarityMax Western ECL Substrate (Bio-Rad, CA, USA) and quantified by ImageJ software (Rawak Software Inc., Germany).
9
Evaluating Protein Expression via Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed to evaluate the levels of relative proteins in cells and tissues. Briefly, the cells and 10 anti‐CitH3 (1:1000; Cat.no.ab5103, Abcam, Cambridge, England, UK), anti‐VEGF (1:1000; Cat.no.sc‐7269, Santa Cruz, Dallas, Texas, USA), anti‐PCNA (1:1000; Cat.no.ab92552, Abcam, Cambridge, England, UK), anti‐MMP2 (1:1000; Cat.no.ab92536, Abcam, Cambridge, England, UK), anti‐MMP9 (1:1000; Cat.no.ab283575, Abcam, Cambridge, England, UK), anti‐FN (1:1000; Cat.no.sc‐8422, Santa Cruz, Dallas, Texas, USA), anti‐E‐cadherin (1:1000; Cat.no.3195S, CST, Boston, Massachusetts, USA), anti‐vimentin (1:1000; Cat.no.sc‐6260, Santa Cruz, Dallas, Texas, USA), anti‐S100A9 (1:1000; Cat.no.58706, Santa Cruz, Dallas, Texas, USA) and anti‐PAD4 antibodies (1:1000; Cat.no.ab214810, Abcam, Cambridge, England, UK). Then the membranes were washed, incubated with secondary antibodies (1:1000; BM6027, Boster, Wuhan, Hubei, China) conjugated with horseradish‐peroxidase and detected by Bio‐Rad assays (733BR4624, Bio‐Rad, Hercules, California, USA). All the experiments were performed thrice.
10
Investigating PopuCATH's Effects on Neutrophil Immunity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The effects of PopuCATH (10 µM) on neutrophil phagocytosis was assayed similar with macrophages phagocytosis as mentioned above. Neutrophils were pre-incubated with PopuCATH (10 µM) for 2 hr, and were incubated with CSFE-loaded S. aureus and E. coli particles at multiplicity of infection 100. At indicated time points, CFSE fluorescence were analysed by flow cytometry (Nijnik et al., 2010 (link)).
For neutrophil extracellular traps (NETs) assay, neutrophils suspended in 2% FBS RPMI 1640 was seeded in an eight well-cover slip chamber (200 μL/well, 1 × 106 cells/mL). PopuCATH (10 µM), PBS, or PMA (100 nM) was added and incubation at 37℃ for 4 hr. Neutrophils were stained with DAPI (Invitrogen, USA) or anti-Cit-H3 (Abcam, USA), respectively. Nuclei and NETs were observed under a confocal microscope ( × 60, Nikon, Japan) (He et al., 2019 (link)).
For neutrophil extracellular traps (NETs) assay, neutrophils suspended in 2% FBS RPMI 1640 was seeded in an eight well-cover slip chamber (200 μL/well, 1 × 106 cells/mL). PopuCATH (10 µM), PBS, or PMA (100 nM) was added and incubation at 37℃ for 4 hr. Neutrophils were stained with DAPI (Invitrogen, USA) or anti-Cit-H3 (Abcam, USA), respectively. Nuclei and NETs were observed under a confocal microscope ( × 60, Nikon, Japan) (He et al., 2019 (link)).
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!