Antibodies were obtained from the following sources: p65 subunit of NF-κB, phospho and total GSK3β (Cell Signaling); PCNA (Santa Cruz); 3-nitrotyrosine (Abcam), actin (Genscript), bromodeoxyuridine (BrdU, Novus Biosciences); DAPI (Invitrogen). TUNEL (Roche Applied Science) was performed according to the manufacturer’s instructions as in 22 (link). Appropriate secondary antibodies for immunohistochemistry and SDS-PAGE were obtained from Molecular Probes and Jackson ImmunoResearch Laboratories. Cetuximab (Dose: 100 μg/day i.p. for 3 days prior to experiments) was a generous gift of Jennifer Grandis (University of Pittsburgh, Pittsburgh, PA). Human Recombinant EGF was from EMD Millipore (400 ng/mL media in vitro, 0.5ng per μL of NEC formula in vivo). Lithium chloride (LiCl) from J.T. Baker (100μM). Lipopolysaccharide (LPS) (Escherichia coli 0111:B4 purified by gel-filtration chromatography, >99% pure) was obtained from Sigma-Aldrich and concentrations used were those that we have demonstrated to be present in mice and humans with NEC (50 μg/mL for cells, 5mg/kg for all mice3 with the exception of the TLR4ΔIEC-OVER mice who are sensitive to LPS and required a decreased dosage (2.5 mg/kg).
Actin
Manufactured by GenScript
Sourced in United Kingdom, United States
Actin is a cytoskeletal protein that plays a crucial role in various cellular processes. It is a globular protein that can polymerize into long, thin filaments known as actin filaments or microfilaments. Actin filaments provide structural support, facilitate cell motility, and are involved in cellular division and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
3 nitrotyrosine, by Abcam (2 mentions)
Phospho and total gsk3β, by Cell Signaling Technology (2 mentions)
Recombinant human egf, by Merck Group (2 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharide lps, by Merck Group (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
β catenin, by Thermo Fisher Scientific (2 mentions)
Cell recovery solution, by BD (2 mentions)
Sds polyacrylamide gel, by Bio-Rad (2 mentions)
Tubulin, by GenScript (2 mentions)
Ir dye 680 anti mouse or, by LI COR (2 mentions)
Ir dye 800 anti rabbit immunoglobulin g igg secondary antibodies, by LI COR (2 mentions)
Tra2β, by Abcam (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
P27kip1, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Boston BioProducts (1 mentions)
Total s6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Phospho c raf, by Cell Signaling Technology (1 mentions)
9 protocols using actin
1
Comprehensive Antibody Protocol for NEC
2
Comprehensive Antibody Protocol for NEC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies were obtained from the following sources: p65 subunit of NF-κB, phospho and total GSK3β (Cell Signaling); PCNA (Santa Cruz); 3-nitrotyrosine (Abcam), actin (Genscript), bromodeoxyuridine (BrdU, Novus Biosciences); DAPI (Invitrogen). TUNEL (Roche Applied Science) was performed according to the manufacturer’s instructions as in 22 (link). Appropriate secondary antibodies for immunohistochemistry and SDS-PAGE were obtained from Molecular Probes and Jackson ImmunoResearch Laboratories. Cetuximab (Dose: 100 μg/day i.p. for 3 days prior to experiments) was a generous gift of Jennifer Grandis (University of Pittsburgh, Pittsburgh, PA). Human Recombinant EGF was from EMD Millipore (400 ng/mL media in vitro, 0.5ng per μL of NEC formula in vivo). Lithium chloride (LiCl) from J.T. Baker (100μM). Lipopolysaccharide (LPS) (Escherichia coli 0111:B4 purified by gel-filtration chromatography, >99% pure) was obtained from Sigma-Aldrich and concentrations used were those that we have demonstrated to be present in mice and humans with NEC (50 μg/mL for cells, 5mg/kg for all mice3 with the exception of the TLR4ΔIEC-OVER mice who are sensitive to LPS and required a decreased dosage (2.5 mg/kg).
3
Comprehensive Cell Signaling Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysates were prepared in RIPA Lysis buffer (Boston Bioproducts), containing protease and phosphatase inhibitors. Protein concentration was determined by BCA protein assay (Pierce) and equal amounts (25 μg) were subjected to SDS-PAGE. Western blot analysis was performed using monoclonal antibodies against CDK1, CDK4, CDK6, cyclin E (Santa Cruz Biotechnology) Cyclin D3, total S6 ribosomal protein, total RB, p27Kip1 (Cell Signaling Technology) and actin (Genscript) and polyclonal antibodies against CDK2, CDK4, p21Waf1/Cip1, pAKT, RSK1/2 (Santa Cruz Biotechnology), Cyclin D1, phospho-RB S807/811, phospho-RB T821, phospho-ERK1/2, total ERK1/2, total AKT, phospho-S6 ribosomal protein, phospho-4E-BP1, total 4E-BP1, total TSC2, phospho-TSC T1462, C-Raf, phospho-C-Raf, MEK1/2, phospho-MEK1/2, phospho-RSK1, E-cadherin, vimentin, N-cadherin, CDK7, phosphor-CDK2 T160 (Cell Signaling Technology) and phospho-TSC S1798 (Abgent).
4
Cytosolic and Nuclear Protein Extraction from Jurkat Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Jurkat cells were washed twice with ice-cold PBS. To extract cytosolic and nuclear protein, Nuclear and Cytoplasmic Protein Extraction Kit was used according to the manufacture's instructions (Beyotime, Haimen, China). Proteins from the extraction were resolved by electrophoresis, transferred to a PVDF membrane, and hybridized with antibodies for strep (GenScript, Nanjing, China, 1:2000), H3 (Abcam, Cambridge, UK, 1:4000), actin (GenScript, Nanjing, China, 1:2000), NFκB P65 (Sangon Biotech, Shanghai, China, 1:500), IKK and IκB (Beyotime, Shanghai, China, 1:1000). Next, the membranes were incubated in secondary antibody, IRDye® 800CW Goat anti-Mouse IgG and IRDye® 680RD Goat anti-Rabbit IgG (both from LI-COR Biosciences, Lincoln, NE) for 2 hours and the specific bands on the membranes were detected using Odyssey® Imaging Systems (LI-COR Biosciences, Lincoln, NE).
5
Western Blot Analysis of DNA Damage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Standard western blotting techniques were used as previously described[17 (link), 21 (link)]. Briefly, cells were pelleted and lysed with Complete Lysis-M buffer (Roche). Whole cell extracts were subjected to SDS-PAGE electrophoresis, transferred to a nitrocellulose membrane, and stained with the following antibodies: p53 (Calbiochem), p53S15 (serine-15), p-ATM (serine-1981), p-BRCA1 (serine-1524), γH2AX (serine-139) (phosphorylated antibodies from Cell Signaling) and Actin (GenScript), followed by the corresponding secondary antibody conjugated with HRP (Santa Cruz).
6
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies against specific proteins were used for Western blot analysis: Actin (A00702, GenScript), CENPW (SC-137988), NCAPG (AP19113a, Abgent), MCM2 (BS1221, Bioworld), PCNA (BS1289, Bioworld), p-ERK (9101, Cell Signaling), ERK (9102, Cell Signaling), p-JNK (9255, Cell Signaling), JNK (9252, Cell Signaling), p-p38 (1229–1, Epitomics), p38 (54419, AnaSpec), p-STAT3 (9134, Cell Signaling) and STAT3 (Sc-482, Santa Cruz, USA). Total protein was extracted from the cells using RIPA buffer as described previously [84] (link). Quantification of total protein lysate was analyzed using a BCA protein assay kit (Pierce Chemical). Protein from each condition (20 μg) was separated with 8 ∼ 12% SDS–PAGE and then blotted onto PVDF membranes (Millipore). The protein-blotted PVDF membranes were further probed with specific antibodies (1:5000 dilution) overnight at 4 °C. The membranes were washed and then reprobed with HRP-conjugated secondary antibodies (1:10,000) at room temperature for 1 hr. The signal on protein-blotted membranes was visualized using ECL (Enhanced Chemiluminescence) reagents (SuperSignal, Pierce Chemical) and autoradiography films (Kodak Rochester, NY).
7
Protein Expression Analysis in 2D and 3D Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3D-grown MCF-10A and MCF-10A MYC-ER cells were washed with PBS (1X) and the Matrigel was dissolved by incubating slides at 4°C in Cell Recovery Solution (BD Biosciences). 2D-grown MDA-MB231 rTTA3 lines and HCC1806-MYC rTTA3 cells were washed with 1xPBS.Cells were lysed in Laemmli buffer (50 mM Tris-HCl pH 6.2, 5% (v/v) β-mercaptoethanol, 10% (v/v) glycerol, 3% (w/v) SDS). Equal amounts of total protein were loaded on a stain-free 12% SDS-polyacrylamide gel (Biorad), transferred onto a nitrocellulose membrane (Millipore) and blocked in 5% (w/v) milk in Tween 20-TBST (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween 20). Blots were incubated with TRA2β (Abcam), SRSF1 (CSHL), SRSF3 (MBL), SRSF7 (MBL), c-MYC (Cell Signaling), Actin (GenScript), Tubulin (GenScript) or β-catenin (ThermoFisher) primary antibodies. IR-Dye 680 anti-mouse or IR-Dye 800 anti-rabbit immunoglobulin G (IgG) secondary antibodies (LI-COR) were used for infrared detection and quantification with a ChemiDoc MP Imaging System (Bio-rad).
8
Protein Expression Analysis in 2D and 3D Cultures
9
Quantifying Immune Cytokine Profiles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytokines (IL-10, IL-17A, IL-12 p70, TGF-β1, IL-6 and IL-23) were measured using a commercially available ELISA kit (Bender: IL-10, IL-17A, IL-12 p70 kits; SABC: IL-23 kit; eBioscience: TGF-β1, IL-6 kits) according to the manufacturer’s instructions. For western blotting, cells were lysed using 0.5 % NP40 lysis buffer and proteins were blotted following standard protocol. Antibodies to RORγt (Abcam) and actin (GenScript Corp) were purchased commercially.
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!