Complete protease and phosstop phosphatase inhibitors

Pieces of BAT were lysed in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 1% SDS; Thermo Fisher Scientific) supplemented with Complete Protease and phosSTOP phosphatase inhibitors (Roche Diagnostics) with the Qiagen TissueLyser II (Qiagen). Protein concentrations were determined using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Western blots were carried out on the Wes system (ProteinSimple) following the manufacturer’s instructions using the primary antibody of tyrosine hydroxylase (TH, 1/250; Ab112; Abcam) and phospho-hormone-sensitive lipase (HSL, Ser563, 1/500; #4139; Cell Signaling). Protein expression levels were normalized to GAPDH (1/1000; #25778; Santa Cruz) housekeeping protein expression. Western blot quantifications were done with Image J software.

Cells were collected and lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) plus Complete protease and PhosSTOP phosphatase inhibitors (Roche Applied Science) on ice for 30 min. Proteins were collected as supernatants after centrifugation at 20,000 g for 15 min at 4 °C. SDS–PAGE was performed using 8–16% or 16% Tris Glycine gels (Invitrogen). Proteins were transferred onto PVDF membranes and detected using standard immunoblotting procedures. To detect phosphorylated PINK1, 8% Tris Glycine gels containing 50 μM Phos-tag acrylamide (Wako chemicals) and 100 μM ZnCl₂ were used [18 (link)]. After electrophoresis, Phos-tag acrylamide gels were washed using transfer buffer with 0.01% SDS and 1 mM EDTA for 20 min and then with transfer buffer containing 0.01% SDS without EDTA for 10 min to remove excess Zn²⁺ before transfer.