Protein Extraction and Immunoblotting

According to previously established procedures, cells and muscle samples were harvested, washed with 1× PBS, and lysed in NP40 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, and 10% glycerol) with protease inhibitor cocktail (Sigma).⁴¹ (link) Proteins were separated in SDS-PAGE, transferred, and immunoblotted with various antibodies. The antibodies used were anti-MyoD (1:1,000; Invitrogen) and anti-β-actin (dilution 1:3,000; Santa Cruz Biotechnology).

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Zhang Z.K., Li J., Guan D., Liang C., Zhuo Z., Liu J., Lu A., Zhang G, & Zhang B.T. (2018). Long Noncoding RNA lncMUMA Reverses Established Skeletal Muscle Atrophy following Mechanical Unloading. Molecular Therapy, 26(11), 2669-2680.

Publication 2018

protease inhibitor cocktail anti-MyoD anti-β-actin

Actin Antibodies Buffer Cells Dilution Edta Glycerol Link proteins Muscle Nacl Np 40 Protease inhibitor Sds page Tris

Corresponding Organization : Hong Kong Baptist University

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Variable analysis

independent variables

Cell and muscle sample harvesting
Washing with 1x PBS
Lysis in NP40 lysis buffer
Addition of protease inhibitor cocktail

dependent variables

Protein expression levels of MyoD and β-actin

control variables

50 mM Tris-HCl
150 mM NaCl
0.1% NP-40
5 mM EDTA
10% glycerol

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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