Two hundred and ninety three cells in four wells of a 24-well plate were transfected with a plasmid encoding firefly luciferase together with an empty plasmid or a plasmid encoding wild-type or mutant PA-X possessing a C-terminal FLAG tag. At 24 h post-transfection, the transfected cells from three of the wells were analyzed for firefly luciferase activity by using the Bright-Glo luciferase assay system (Promega). Data are shown as the average luminescence ± standard deviation (n = 3). To analyze the expression of PA-X, the transfected cells in the remaining well were lysed in SDS sample buffer at 24 h post-transfection. The cell lysates were sonicated, incubated for 10 min at 95°C, and then loaded onto an Any KD Mini-PROTEAN TGX Gel (Bio-Rad). Separated proteins were transferred to Immobilon-P PVDF membrane (Millipore) and detected by using anti-DYKDDDDK (FLAG) tag antibody clone 1E6 (Wako) or anti-β-actin antibody clone AC-74 (Sigma), followed by a sheep anti-mouse IgG-HRP secondary antibody (GE Healthcare).
Anti β actin antibody clone ac 74
Manufactured by Merck Group
Sourced in United States
The Anti-β-actin antibody clone AC-74 is a mouse monoclonal antibody that recognizes the β-actin protein. It is commonly used in various laboratory applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and quantify the expression of β-actin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p pvdf membrane, by Merck Group (2 mentions)
Any kd mini protean tgx gel, by Bio-Rad (2 mentions)
Anti dykddddk flag tag antibody clone 1e6, by Fujifilm (2 mentions)
Bright glo luciferase assay system, by Promega (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Sheep anti mouse igg hrp secondary antibody, by GE Healthcare (1 mentions)
Siinfekl peptide, by AnaSpec (1 mentions)
Novex ecl hrp chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris gel, by Bio-Rad (1 mentions)
Sheep anti mouse igg hrp, by GE Healthcare (1 mentions)
Luminata forte, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti yap, by Cell Signaling Technology (1 mentions)
Anti phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Manganese 3 tetrakis 4 benzoic acid porphyrin chloride mntbap, by Merck Group (1 mentions)
Annexin 5 apoptosis detection kit, by BD (1 mentions)
8 protocols using anti β actin antibody clone ac 74
1
Luciferase Assay for PA-X Expression
2
Multiparametric Immune Cell Analysis
3
Quantifying Protein Expression via SDS-PAGE and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 15 μg of protein from each sample on a 4–12% NuPAGE Bis-Tris Gel (Bio-Rad, USA). The proteins were blotted onto polyvinylidene difluoride (PVDF) membranes, blocked with 5% dry milk and incubated at 4°C overnight with anti-HE antibody (clone 8D2/E9; GrupoBios, Bios-Chile) diluted 1:2000 or anti-β-actin antibody (clone AC-74, Sigma-Aldrich) diluted 1:500, followed by a 1 h incubation with the secondary antibody [rabbit-anti-mouse horseradish peroxidase (HRP), Invitrogen, USA] at a dilution of 1:20000. The blots were developed using Novex® ECL HRP Chemiluminescent Substrate (Invitrogen, USA) and exposed to Carestream Kodak Biomax Light film (sigma Aldrich, USA). The films were scanned and used for expression analysis with ImageJ 1.43 μ software.
4
Measuring PA-X Mediated Host Shutoff
5
Subcellular Proteome Extraction and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed with ProteoExtract Subcellular Proteome Extraction kit (Calbiochem, San Diego, CA, USA) and extracted cytoplasm and nuclear fraction following the manufacturer’s instructions, and the cell lysates were separated on SDS-polyacrylamide gels and blotted onto PVDF membranes (GE Healthcare, Buckinghamshire, UK).The membranes were reacted overnight at 4 °C with each primary antibody: anti-phospho-ERK1/2 (#9101), anti-ERK1/2 (#9102), anti-phospho-Akt (#9271), anti-Akt (#9272), anti-phospho-NF-κB p65 (#3031), anti-NF-κB p65 (#3034), anti-YAP (#8418) (Cell Signaling Technology, Beverly, MA, USA), anti-RhoA (F-1), anti-RhoB (C-5), anti-RhoC (C-16), anti-RhoE (clone 4), anti-RHAMM (C-9), anti-CD44 (F-4), anti-CXCR4 (4G10), anti-CCR7 (A-19), lamin A/C antibody (H-110) (Santa Cruz Biotechnologies, CA, USA), and anti-β-actin antibody (clone AC-74) (Sigma). The membranes were reacted with horseradish peroxidase-coupled sheep anti-rabbit IgG (GE Healthcare) for 1 h and were subsequently visualized using Luminata Forte (Merck Millipore, Nottingham, UK).
6
Immunophenotyping of T Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against CD3 (clone 145-2C11), CD28 (clone 37.51), CD25 (clone PC61), CD28 (clone CD28.2), CD44 (clone IM7), and CD69 (clone H1.2F3) were obtained from Becton Dickinson Biosciences (BD Biosciences, San Jose, CA). Antibodies against Sca-1 (clone D7) CD62L (clone MEL-14), CCR7 (clone 4B12), CD122 (clone 5H4), and CD127 (clone SB/199) were obtained from eBioscience. Anti-phospho-4E-BP1 (clone 236B4), anti-4E-BP1 (clone 53H11), and anti-TSC2 (D93F12) antibodies were obtained from Cell Signaling Technology (Danvers, MA). Anti-β-actin antibody (clone AC-74) was obtained from Sigma-Aldrich (St. Louis, MO). Nx-hydroxy-nor-Arginine (N-NOHA), L-NG-Monomethylarginine (L-NMMA), and Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) were obtained from EMD Millipore (Calbiochem, Gibbstown, NJ). Induction of apoptosis was tested using annexin V Apoptosis Detection Kit (BD Biosciences). Results were expressed as the percentage of annexin V+ cells within CD8+ cells.
7
Foxp3 Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sorted cells were lysed in Radioimmunoprecipitation assay buffer (RIPA) buffer (150 mM NaCl, 10 mM Tris-Cl, pH 7.4, 5 mM EDTA, 1% Triton X100, 0.1% SDS, 0.5% sodium deoxycholate). Proteins (10 to 50 µg) were separated and membranes were incubated overnight at 4°C with anti-mouse Foxp3 purified antibodies and 1 h at room temperature with anti-β actin antibody (clone AC-74, Sigma Aldrich). Immunoreactive bands were visualized by enhanced chemiluminesence (Amersham).
8
Western Blot Analysis of IL18RAP Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysate was obtained by lysing neutrophils with lysis buffer (Cell Signaling Technology, Danvers, MA, USA) of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, 1 mM PMSF (Sigma-Aldrich, Steinheim, Germany) and protease inhibitor cocktail. Protein from each sample (30 μg) was separated by SDS-PAGE and transferred to PVDF membrane (GE Healthcare Life Science, Freiburg, Germany), followed by incubation with anti-IL18RAP monoclonal antibody (clone 290, Invitrogen, Rockford, IL, USA) or with anti-β-actin antibody (clone AC-74, Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C. After incubation with HRP-conjugated antirabbit or antimouse IgG (Santa Cruz, Dallas, TX, USA), membranes were exposed to enhanced chemiluminescence substrate. Signals were detected by a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Densitometry reading of the blots was analysed by Image Lab Software (Bio-Rad Laboratories, Hercules, CA, USA) and IL18RAP signal intensity was normalized with the β-actin level in the corresponding sample.
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!