Total cell lysates from human RMS cell lines and human myoblasts were obtained following lysis in RIPA lysis buffer supplemented with protease inhibitors (Roche). Western blot analysis was performed similar to Ignatius et. al., 2017 (27 (link)). Membranes were developed using an ECL reagent (Western Lightning Plus ECL, PerkinElmer; or sensitive SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific). Membranes were stripped, rinsed, and re-probed with the respective internal control antibodies. List of primary and secondary antibodies is included in supplementary data (Supplemental Table 1 ). Western blots were quantified using ImageJ software, normalizing proteins of interest to their respective β-tubulin expression and the control (either hSKMC or Scr of the same time point). If control expression was zero, quantifications shown are shown as a ratio of the protein of interest expression to its respective β-tubulin expression.
Protocol detail
Western Blot Analysis of RMS Cell Lines
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protease inhibitors
Western Lightning Plus ECL
sensitive SuperSignal West Femto Maximum Sensitivity Substrate
Antibodies
Buffer
Cell lines
Human
Membranes
Myoblasts
Protease inhibitors
Protein a
Proteins
Ripa
Sensitivity
Tubulin
Western blots
Corresponding Organization :
Other organizations : The University of Texas Health Science Center at Houston, The University of Texas Health Science Center at San Antonio, Wenzhou Medical University, Nationwide Children's Hospital, Case Western Reserve University, Bambino Gesù Children's Hospital, The Ohio State University, University of Washington, Center for Cancer Research
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Variable analysis
independent variables
- Cell line type (human RMS cell lines vs. human myoblasts)
- Protein expression levels (normalized to β-tubulin and control)
- RIPA lysis buffer supplemented with protease inhibitors (Roche)
- Western blot analysis procedure (similar to Ignatius et al., 2017)
- Membrane development using ECL reagents (Western Lightning Plus ECL, PerkinElmer; or SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific)
- Stripping, rinsing, and re-probing of membranes with internal control antibodies
- Quantification using ImageJ software
- Positive control: hSKMC (human skeletal muscle cells)
- Negative control: Scr (Scrambled control) of the same time point
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