Western Blot Analysis of MCPIP1 and NF-κB Signaling

Treated cells were lysed using the Mammalian Cell Lysis kit (Sigma-Aldrich). Equal amounts of the proteins were electrophoresed in a sodium dodecyl sulphate-polyacrylamide gel (12%) under reducing conditions followed by transfer to polyvinylidene fluoride membranes. The blots were blocked with 5% non-fat dry milk in PBS. The western blots were then probed with antibodies recognizing the MCPIP1 (1:1,000, Santa Cruz), β-actin (1:1,000, Sigma-Aldrich) p65 NF-κB (1:200, Cell Signalling), β-catenin (1:200, Cell Signalling), Lamin B (1:1,000, Santa Cruz) The secondary antibodies were horseradish peroxidase-conjugated to goat anti mouse/rabbit IgG (1:5,000) as described previously43 (link). Full blots can be found in Supplementary Fig. 14.

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Yao H., Ma R., Yang L., Hu G., Chen X., Duan M., Kook Y., Niu F., Liao K., Fu M., Hu G., Kolattukudy P, & Buch S. (2014). MiR-9 promotes microglial activation by targeting MCPIP1. Nature Communications, 5, 4386.

Publication 2014

Mammalian Cell Lysis kit MCPIP1 β-actin p65 NF-κB β-catenin Lamin B

Actin Anti igg Antibodies Cell Goat Horseradish peroxidase Lamin b Mammalian Membranes Milk Mouse Nf κb Polyacrylamide gel Polyvinylidene fluoride Proteins Rabbit Sodium dodecyl sulphate Western blots β catenin

Corresponding Organization :

Other organizations : Southeast University, University of Nebraska Medical Center, Nanjing Medical University, Jilin University, University of Missouri–Kansas City, University of Central Florida

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Variable analysis

independent variables

Treatment of cells

dependent variables

Protein expression levels of MCPIP1, p65 NF-κB, β-catenin, and Lamin B

control variables

Protein loading (equal amounts of proteins were electrophoresed)
Loading control (β-actin)

controls

Positive control: Not specified
Negative control: Not specified

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