Phosphatase inhibitor cocktail set 4

Cells were washed with PBS and lysed with radio-immunoprecipitation assay buffer (R0278, Sigma) supplemented with phosphatase inhibitor cocktail set IV (1:50, 524628; Merck Millipore, Billerica, MA, USA) and PMSF protease inhibitors (1 nM). Cell lysates were centrifuged at 14,000×g for 30 min at 4 °C, and a Bradford assay was performed. A protein suspension (30 µL) containing 30 µg total protein and 100 µL 0.1% L-DOPA in PBS was mixed in a 96-well plate. After incubation at 37℃ for 1 h, dopachrome formation was measured at 475 nm. Tyrosinase activity was calculated as the percentage of the control.

Frozen tissue samples were weighed, and the appropriate amount of RIPA lysis buffer (1 ml/100 μg of tissue, 150 mM sodium chloride, 1.0% sodium dodecyl sulfate, 50 mM Tris pH 8.0, protease inhibitor cocktail set III (1 : 200, Merck, Kilsyth, VIC, Australia), and phosphatase inhibitor cocktail set IV (1 : 50, Merck, Kilsyth, VIC, Australia)) was added to the samples before homogenization with a hand-held homogenizer. Homogenized samples were then left on ice for 10 minutes before rotating slowly for an hour at 4°C. Samples were then centrifuged for 15 mins at 14000 ×g, at 4°C. Protein containing supernatant was carefully isolated, and a bicinchoninic acid (BCA) protein assay was performed (Thermo Scientific, Rockford, IL, USA).

Samples were lysed for 10 min on ice in lysis buffer containing 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10 mM EDTA, 100 mM NaF, 1% (v/v) Nonidet P-40, 10 mM Na₃VO₄, 2 mM PMSF, 1% (v/v) protease inhibitor cocktail set V (Merck Millipore) and 1% (v/v) phosphatase inhibitor cocktail set IV (Merck Millipore). Total protein concentration of lysates was quantified using a DC Protein Assay (BioRad) and 100 μg of each sample labelled using TMTsixplex reagents according to manufacturer’s instructions (Thermo Scientific). The labelled samples were pooled and then subjected to phospho-peptide enrichment using a TiO₂-based enrichment kit, according to the manufacturer’s instructions (Pierce). The phospho-enriched sample was then analysed by RP nano-LC MSMS using an LTQ-Orbitrap Velos mass spectrometer.