Cells were seeded in 6-well plates as described above for 48 h but at a density of 1 × 106 in 3 mL culture medium. Following this period, the culture medium was replaced with 2 mL fresh culture medium for 2 h. Cells were lysed with radioimmunoprecipitation assay lysis buffer supplemented with Halts protease and phosphatase inhibitors (Thermo Fisher Scientific). Total protein was quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of total cell lysate (30 mg per well) were subjected to sodium dodecyl sulfate electrophoresis on 4 to 12% Bis-Tris gels (Thermo Fisher Scientific). Gels were run at a constant voltage of 100 mV (PowerPac 3000, Bio-Rad) and transferred onto a nitrocellulose membrane with the iBlot2 Transfer System (Thermo Fisher Scientific) as per manufacturer’s instructions. Immunoblotting was completed for GAPDH, IL-17RA, TRAF6 (all Cell Signaling Technology), and IL-17RC (Abcam). A Li-Cor Azure C500 imager was used to visualize immunoblots, and ImageJ software (NIH) was used for quantification.
Il 17ra
Manufactured by Cell Signaling Technology
IL-17RA is a cell surface receptor that binds the cytokine IL-17A. It is involved in the regulation of inflammatory and immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Traf6, by Cell Signaling Technology (1 mentions)
Halt s protease and phosphatase inhibitors, by Thermo Fisher Scientific (1 mentions)
Powerpac 3000, by Bio-Rad (1 mentions)
4 to 12 bis tris gels, by Thermo Fisher Scientific (1 mentions)
Iblot2 transfer system, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Strep tag 2, by Qiagen (1 mentions)
2 protocols using il 17ra
1
Quantification of Signaling Proteins
2
Affinity Purification of Strep-Tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected and treated HEK293T cells were pelleted and washed in cold D-PBS and later resuspended in Flag-IP Buffer (50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% NP-40) with 1x HALT (ThermoFisher Scientific, 78429), incubated with buffer for 15 min on ice then centrifuged at 13,000 rpm for 5 min. The supernatant was collected and 1 mg of protein was used for immunoprecipitation (IP) with 100 μl of Streptactin Sepharose (IBA, 2-1201-010) on a rotor overnight at 4°C. Immunoprecipitates were washed five times with Flag-IP buffer and eluted with 1x Buffer E (100 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 2.5 mM Desthiobiotin). Eluate was diluted with 1x-NuPAGE (ThermoFisher Scientific, #NP0008) LDS Sample Buffer with 2.5% β-Mercaptoethanol and blotted for targeted antibodies. Antibodies used were Strep tag II (Qiagen, #34850), B-Actin (Sigma, #A5316), and IL17RA (Cell Signaling, #12661S).
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!