Pierce protease and phosphatase inhibitor mini tablet

At the end of the perfusions the hearts were homogenized using a modified protocol described previously [22 (link)]. Briefly, all the procedures were conducted at 4 °C to maintain the integrity of mitochondrial proteins. The hearts were immersed in the homogenization buffer (in 50 ml: 10 mM Tris HCl, 5 tablets of protease inhibitor (Pierce Protease and Phosphatase Inhibitor Mini Tablets, ThermoFisher, catalog A32959), and 250 mmol/l sucrose pH 7.0). The hearts were then chopped into small pieces with small scissors and homogenized with a teflon pestle in a glass tube. Tissue homogenate was centrifuged at 800×g for 10 min to pull down tissue debris while the supernatant was collected and centrifuged at 10,000×g for 20 min to separate the mitochondrial fraction as a pellet. The supernatant from the second centrifugation was further centrifuged at 105,000×g for 60 min to separate microsomes from the cytosolic fraction. For subsequent investigations, mitochondrial and cytosolic fractions were used. Protein concentration in each fraction was measured using Bradford assay (BioRad, USA).

AICAR was purchased from Selleck Chemicals (Lot #7B/237853, Cat #1802) for in vitro and MedchemExpress (Lot #97416, Cat #HY-13417) for in vivo applications. VX-509 (Lot #S754101, Cat #S7541), ABT-702 (Cat #S6619), and osimertinib (Cat #S7297) were purchased from Selleck Chemicals. Pierce protease and phosphatase inhibitor mini tablets (Lot #WD319834, Cat #A32959) were from Thermo Fisher Scientific and utilised for protein extraction.

The lumbar-sacral portion of the spinal cord (approximately 1 cm from the distal end) was flash frozen and cryopulverized in the presence of liquid nitrogen and divided equally into two aliquots for RNA extraction or protein extraction. Protein extraction was done by homogenization in RIPA buffer containing protease and phosphatase inhibitors (Pierce Protease and Phosphatase Inhibitor Mini Tablets, ThermoScientific). 30 µg of spinal cord lysate was separated in a 4–20% Tris–glycine gel (Novex, Invitrogen), proteins transferred to PVDF membranes and blocked in 0.5% casein. Membranes were incubated in appropriate primary antibodies (anti V5, 1:1000, ThermoFisher # R960; GAPDH, 1:1000, Abcam # ab181602) and secondary antibodies conjugated to IRDye (1:20,000; Li-Cor Biotechnology) diluted in 0.5% casein and protein bands were detected using the multiplex Li-Cor Odyssey Infrared Imaging system (Li-Cor Biotechnology, Lincoln, NE, USA).