Western Blot Analysis of Protein Expression

Protein expression was analysed using western blotting.^{35 (link)} Cells/sciatic nerves were lysed in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher); sciatic nerves were sonicated into lysis buffer using a Q500 sonicator (Thermo Fisher). Lysates were run on sodium dodecyl sulphate–polyacrylamide gels (Bio-Rad), transferred onto polyvinylidene-fluoride (PVDF) membranes (Cytiva), blocked in 5% bovine serum albumin (BSA), incubated with primary antibodies in BSA overnight at 4°C and then horse radish peroxidase (HRP)-conjugated secondary antibodies in 5% BSA for 1 h at room temperature. Blots were visualized using Pierce™ enhanced chemiluminescence (ECL) (Thermo Fisher) on a PXi developer (Syngene), quantified by densitometry and normalized to glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) or vinculin loading controls using ImageJ. In Figs 2, 5E and 6E, blots shown are representative of independent biological repeats; Fig. 5L shows collated blots from the same three paired biological repeats. Figure 7D shows three paired biological repeats on the same blot. Figure 8C and E show representative blots of technical repeats.

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Laraba L., Hillson L., de Guibert J.G., Hewitt A., Jaques M.R., Tang T.T., Post L., Ercolano E., Rai G., Yang S.M., Jagger D.J., Woznica W., Edwards P., Shivane A.G., Hanemann C.O, & Parkinson D.B. (2022). Inhibition of YAP/TAZ-driven TEAD activity prevents growth of NF2-null schwannoma and meningioma. Brain, 146(4), 1697-1713.

Publication 2022

RIPA) buffer sodium dodecyl sulphate–polyacrylamide gels PVDF) membranes Pierce™ enhanced chemiluminescence (ECL)

Albumin in serum Antibodies Biological Bovine Bovine serum albumin Buffer Cells Chemiluminescence Densitometry Glyceraldehyde dehydrogenase Horse radish peroxidase Membranes Phosphatase Polyacrylamide gels Polyvinylidene fluoride Protein Radioimmunoprecipitation assay Sciatic nerves Sodium dodecyl sulphate Vinculin

Corresponding Organization : University of Plymouth

Other organizations : University of Bath, National Center for Advancing Translational Sciences, National Institutes of Health, University College London, University Hospitals Plymouth NHS Trust

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Variable analysis

independent variables

Protein expression analysis using western blotting

dependent variables

Protein expression levels

control variables

Cells/sciatic nerves were lysed in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher)
Sciatic nerves were sonicated into lysis buffer using a Q500 sonicator (Thermo Fisher)
Lysates were run on sodium dodecyl sulphate–polyacrylamide gels (Bio-Rad)
Transferred onto polyvinylidene-fluoride (PVDF) membranes (Cytiva)
Blocked in 5% bovine serum albumin (BSA)
Incubated with primary antibodies in BSA overnight at 4°C
Incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies in 5% BSA for 1 h at room temperature
Blots were visualized using Pierce™ enhanced chemiluminescence (ECL) (Thermo Fisher) on a PXi developer (Syngene)
Quantified by densitometry and normalized to glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) or vinculin loading controls using ImageJ

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