Protease and protein phosphatase inhibitors

Whole-cell lysates were prepared in RIPA lysis buffer in the presence of protease and protein phosphatase inhibitors (Thermo Fisher) as previously described [15 (link)]. Lysates were vortexed every 10 min for 1 h at 4 °C and centrifuged at 15,000× g rpm. Protein concentrations of the supernatants were determined using the BCA assay kit as per the manufacturer’s instructions (Thermo Fisher).

Whole cell lysates were prepared in RIPA Lysis buffer in the presence of protease and protein phosphatase inhibitors (Thermo Fisher). Lysates were vortexed every 10 minutes for 1 hour at 4°C, and centrifuging at 15000 rpm for 5 minutes. Protein concentrations of the supernatants were determined using the BCA assay kit as per manufacturer’s instructions (Thermo Fisher). For western blotting analysis, equal amounts of proteins were heated to 95°C, separated on 4–15% SDS-polyacrylamide gels (Bio-Rad, Australia) and wet transferred to PVDF at 200 mV for 1 hour at 4°C (Polyvinylidene Difluoride) membranes (Thermo Fisher). The membrane was then blocked 2 hours in TBST (50 mmol/L Tris-Cl, pH 7.6, 150 mmol/L NaCl and 0.1% Tween 20) containing 5% (w/v) casein, and then incubated with the appropriate primary antibody as indicated overnight at 4°C. The membrane was washed four times with TBST, then incubated with HRP-conjugated secondary antibody for 1 hour. The membrane was again washed four times with TBST, and the blots were developed using SuperSignal West Femto kit [25 (link)] (Thermo Fisher). All phosho-immunoreactive species, Akt, ERK, SHP were normalized against total Akt, ERK or SHP. Immunoreactive phospho-tyrosine was normalized to GAPDH.

Whole cell protein lysates were prepared in RIPA Lysis buffer in the presence of protease and protein phosphatase inhibitors (Thermo Fisher) as previously described [17 (link)]. Briefly, whole cell lysates were vortexed every 10 min for 1 h on ice and centrifuged at 15,000 rpm for 5 min. The protein concentrations of the supernatants were determined by a BCA assay kit as per manufacturer’s instructions (Thermo Fisher).