Fractionation and Immunoblotting of Cellular Proteins

Protein extraction was carried out as previously described.^{14 (link)} Fractionation of nuclear and cytoplasmic protein was carried out using the NE-PER^TM nuclear and cytoplasmic fraction reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Fifteen micrograms of nuclear protein lysates and equivalent amount of the cytoplasmic protein lysates were separated on 4–15% Precast PAGE gels (Bio-Rad) and electrotransferred to nitrocellulose membranes. The membranes were probed with antibodies against PTPN14 (Sigma-Aldrich, NSW, Australia), YAP (clone D8H1X, Cell Signaling, MA, USA), Taz (clone V386, Cell Signaling), stathmin (BD Bioscience, Victoria, Australia), Topoisomerase I (Novus Biologicals, CO, USA) as a control for nuclear fraction and GAPDH (clone 6C5, Abcam, Victoria, Australia) as a control for equal loading. Proteins were detected by ECL Plus (Pierce, Thermo Fisher Scientific) and membranes were either scanned using the Typhoon (GE Healthcare) or exposed to the film. Densitometry analysis was carried out as previously described.^{15 (link)}