Western Blot Analysis of miRNA Targets

Western blot analysis of miRNA target proteins was performed according to the previous report [35 (link)]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted using a Mini-PROTEIN^® System (Bio-Rad, Hercules, CA., USA) and a 12% gel according to the manufacturer’s protocol. Proteins were transferred to a nitrocellulose blotting membrane and probed overnight at 4 °C with primary antibodies (1:1000, pSmad 2, Smad 2, pSmad 3, Smad 3, and Smad 4, Bioss; Id1, Santa Cruz; β-actin, Sigma-Aldrich) followed by 1 h at RT by an HRP-conjugated secondary antibody (1:2000, rabbit anti-mouse HRP and goat anti-rabbit HRP, Santa Cruz). Immunolabeled proteins were detected by incubation with enhanced chemiluminescence (ECL) substrate, followed by exposure of the membrane to autoradiography film.

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Han S.R., Kang Y.H., Jeon H., Lee S., Park S.J., Song D.Y., Min S.S., Yoo S.M., Lee M.S, & Lee S.H. (2020). Differential Expression of miRNAs and Behavioral Change in the Cuprizone-Induced Demyelination Mouse Model. International Journal of Molecular Sciences, 21(2), 646.

Publication 2020

Mini-PROTEIN® System β-actin rabbit anti-mouse HRP goat anti-rabbit HRP

Actin Antibodies Antibody Autoradiography Chemiluminescence Goat Membrane Mirna Mouse Nitrocellulose Proteins Proteins target Rabbit Sds page Smad 3 Smad 4 Western blot analysis

Corresponding Organization : Eulji University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of miRNA target proteins (pSmad 2, Smad 2, pSmad 3, Smad 3, Smad 4, Id1, β-actin)

control variables

SDS-PAGE run using a Mini-PROTEIN® System (Bio-Rad) and a 12% gel according to the manufacturer's protocol
Proteins transferred to a nitrocellulose blotting membrane
Probing with primary antibodies (1:1000 dilution) overnight at 4 °C
Probing with HRP-conjugated secondary antibodies (1:2000 dilution) for 1 h at room temperature
Detecting immunolabeled proteins using enhanced chemiluminescence (ECL) substrate and autoradiography film

controls

Positive control: None explicitly mentioned
Negative control: None explicitly mentioned

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