Immunoprecipitation of RAW264.7 Cell Lysates

Immunoprecipitation was performed as described previously (21 (link)). Briefly, RAW264.7 lysates were prepared in immunoprecipitation lysis buffer (20 mmol/L Tris-Cl, pH 8.0, 10 mmol/L NaCl, 1 mmol/L EDTA, 0.1% NP-40) containing a protease inhibitor cocktail (Sigma). Two micrograms of cell extracts were precleared with 50 µl of protein A/G-agarose (Thermo Fisher) at 4°C for 2 h, and the supernatant was incubated with the corresponding antibodies with gentle shaking at 4°C overnight, followed by the addition of 20 µl of protein A/G-agarose for another 1 h. The beads were washed and then resuspended in 30 µl of loading buffer and boiled for 5 min, followed by Western blot detection.

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Li R., Sun N., Chen X., Li X., Zhao J., Cheng W., Hua H., Fukatsu M., Mori H., Takahashi H., Ohkawara H., Fukami M., Okamoto M., Hamazaki Y., Zheng K., Yang J, & Ikezoe T. (2021). JAK2V617F Mutation Promoted IL-6 Production and Glycolysis via Mediating PKM1 Stabilization in Macrophages. Frontiers in Immunology, 11, 589048.

Publication 2020

protease inhibitor cocktail protein A/G-agarose

Agarose Antibodies Buffer Cell extracts Edta G protein Immunoprecipitation Nacl Np 40 Protease inhibitor Protein a Protein g Tris Western blot

Corresponding Organization : Fukushima Medical University

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Variable analysis

independent variables

Immunoprecipitation was performed as described previously (21 (link)).

dependent variables

Western blot detection

control variables

RAW264.7 lysates were prepared in immunoprecipitation lysis buffer (20 mmol/L Tris-Cl, pH 8.0, 10 mmol/L NaCl, 1 mmol/L EDTA, 0.1% NP-40) containing a protease inhibitor cocktail (Sigma)
Two micrograms of cell extracts were precleared with 50 µl of protein A/G-agarose (Thermo Fisher) at 4°C for 2 h
The supernatant was incubated with the corresponding antibodies with gentle shaking at 4°C overnight
The beads were washed and then resuspended in 30 µl of loading buffer and boiled for 5 min

positive controls

No positive controls were explicitly mentioned.

negative controls

No negative controls were explicitly mentioned.

Annotations

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