Pre cast page gels

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)}

Cell lines were harvested during active growth (0.4E6 – 0.6E6 cells/mL) and lysed in RIPA buffer supplemented with protease inhibitor cocktail (Halt). Lysates were centrifuged at 16,000 xg for 20 min at 4 °C, and total protein concentration was quantified by Pierce BCA Assay (Thermo). SDS loading buffer was added to 50 μg total protein, and samples were boiled at 95 °C for 5 min. Samples were loaded on 4–15% pre-cast PAGE gels (Bio-Rad), run at 85 V in running buffer, and transferred to a PVDF membrane (Bio-Rad). Membranes were incubated for 1 hour in blocking buffer (1% (w/v) BSA in 0.1% TBS-T), followed by an overnight incubation at 4 °C in blocking buffer containing primary antibody. Membranes were washed for 10 min 0.1% TBS-T three times, followed by a 1 hr incubation with secondary antibody with rotation at room temperature and protected from light. Membranes were washed for 10 min 0.1% TBS-T three times and imaged using an LI-COR Odyssey DLx.
Antibodies used for Western blotting include rabbit anti-IGHMBP2 at 1:500 (Proteintech, 23945–1-AP), rabbit anti-β-actin conjugated to AlexaFluor680 (Abcam); rabbit anti-eIF2α at 1:1,000; rabbit anti-eIF2α phospho-S51 at 1:1,000. Membranes were incubated with goat anti-rabbit 800 CW secondary antibody (LI-COR, 926–32211) at 1:10,000.

Cell lines were harvested during active growth (0.4 × 10⁶–0.6 × 10⁶ cells/ml) and lysed in RIPA buffer supplemented with protease inhibitor cocktail (Halt). Lysates were centrifuged at 16,000g for 20 min at 4°C, and total protein concentration was quantified by Pierce BCA Assay (Thermo Fisher Scientific). SDS loading buffer was added to 50 μg total protein, and samples were boiled at 95°C for 5 min. Samples were loaded on 4–15% pre-cast PAGE gels (Bio-Rad), run at 85 V in running buffer, and transferred to a PVDF membrane (Bio-Rad). Membranes were incubated for 1 h in blocking buffer (1% [wt/vol] BSA in 0.1% TBS-T), followed by an overnight incubation at 4°C in blocking buffer containing primary antibody. Membranes were washed for 10 min with 0.1% TBS-T three times, followed by 1 h incubation with secondary antibody with rotation at RT and protected from light. Membranes were washed for 10 min 0.1% TBS-T three times and imaged using a LI-COR Odyssey DLx.
Antibodies used for Western blotting include rabbit anti-IGHMBP2 at 1:500 (23945-1-AP; Proteintech), rabbit anti-β-actin conjugated to Alexa Fluor 680 (Abcam); rabbit anti-eIF2α at 1:1,000; rabbit anti-eIF2α phospho-S51 at 1:1,000. Membranes were incubated with goat anti-rabbit 800 CW secondary antibody (926-32211; LI-COR) at 1:10,000.