Previously described procedures were used [21 (link), 22 (link)]. Briefly, protein samples (50 μg) were separated using NuPAGE 4–12% Bis-Tris gels (Life Technologies, Carlsbad, CA, USA). Proteins were transferred to polyvinylidene difluoride membranes, which were then blotted with primary antibodies overnight and visualised using ECL Plus substrate (Thermo Scientific, Waltham, MA, USA). The density of the bands was quantified using Quantity One 1-D Analysis Software (Bio-Rad Laboratories, Hercules, CA, USA). Trizol (Life Technologies) was used to isolate total mRNA. Relative PCR quantification was performed using a Stratagene Mx3000P (Agilent Technologies, Santa Clara, CA, USA). Expression data were normalised to the amount of 18S rRNA.
Protocol detail
Western Blot and qPCR Analysis
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Corresponding Organization :
Other organizations : University of California, San Diego, University of Colorado Denver
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Variable analysis
independent variables
- Previously described procedures
- Protein expression levels
- MRNA expression levels
- Protein samples (50 μg)
- NuPAGE 4–12% Bis-Tris gels
- Polyvinylidene difluoride membranes
- Primary antibodies
- ECL Plus substrate
- Trizol for mRNA isolation
- Stratagene Mx3000P for relative PCR quantification
- 18S rRNA for expression data normalization
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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