Protein extraction

Protein samples were prepared as previously described [75]. Briefly, the blade samples were frozen using liquid nitrogen and then ground with a precooled pestle and mortar. A mixture of TCA/acetone (1:9) was then added five times to the powder and blended well by vortexing. The blended mix was then kept at − 20 °C for 4 h and centrifuged at 4 °C for 40 min at 6000×g. The supernatant was then removed and the pellet was rinsed three times using the addition of pre-cooled acetone. The precipitate was then air-dried; SDT buffer (approximately 30 (v/v)) was then mixed with 20–30 mg of powder, blended, and the mixture boiled for 5 min. To ensure efficient extraction, following sonication, the lysate was boiled again for 15 min, and then centrifuged at 14000×g for 40 min at 4 °C. The supernatant was collected, and filtered with a 0.22 μm size filter and the protein content quantified with the BCA Protein Assay Kit (Bio-Rad, USA). Finally, the sample was stored at − 80 °C before further analysis.