Protease and phosphatase inhibitor cocktail

Superoxide dismutase activity was performed as described previously (Tsang et al., 2014 ) with minor modification. Briefly, yeast cells were washed with phosphate buffer (PB) (0.05 M KH₂PO₄ and K₂HPO₄, pH 7.8), and lysed with glass beads by vortexing in PB supplemented with 0.1% Triton X-100, and protease and phosphatase inhibitor cocktails (Roche). 1–10 µg proteins were separated in 12% native PAGE gel for SOD1 and SOD2 activities, respectively. Mammalian cells were washed three times in phosphate-buffered saline (PBS) (KCl 2.7 mM, KH₂PO₄, 1.5 mM, NaHPO₄ 8 mM and NaCl 136.9 mM, pH 7.0), lysed in 50 mM phosphatase buffer (pH 7.8) containing protease and phosphatase inhibitor cocktail (Roche) with a Bioruptor (Diagenode) (30 seconds on and 30 seconds off at midpower) for 10 min. Forty to eighty µg proteins were separated in 12% native PAGE gel. Native PAGE gels were stained with 2.43 mM nitro blue tetrazolium chloride (Sigma), 0.14 M riboflavin-5’-phosphate (Sigma) and 28 mM TEMED (Bio-Rad) for 20 min at room temperature in darkness. To visualize SOD1 and SOD2 activities, gels were rinsed with water twice and placed on a light box for 60 min. SOD1 and SOD2 bands were identified either by sodium cyanide treatment in the gel staining step (Weydert and Cullen, 2010 (link)), by genetic deletion (yeast) or siRNA mediated knockdown (mammalian cells).

Cells were lysed with a buffer containing 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 1% NP-40 supplemented with protease and phosphatase inhibitor cocktails (Roche). Cell debris were removed by centrifugation at 16,000g for 15 min (4°C). Thymus was smashed and homogenized in 1 ml of lysis buffer 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 1% NP-40 supplemented with protease and phosphatase inhibitor cocktails (Roche) using a TissueLyser (full speed for 20 min; Qiagen). The tissue was then maintained in constant agitation for 30 min at 4°C. Sample was centrifuged for 20 min at 13,000g at 4°C, and supernatant was collected. Both cell and thymus proteins were quantified using a bicinchoninic acid assay (Euroclone). Subsequently, 50 μg of cell lysates was run on an 8% polyacrylamide gel, and SDS-polyacrylamide gel electrophoresis was performed following standard procedures. After protein transfer, nitrocellulose membranes (Thermo Fisher Scientific) were incubated with anti-human/mouse ITPKB (ProteinTech), anti-human/mouse GAPDH [CST (Cell Signaling Technology), 14C10], or anti-human/mouse vinculin (E1E9V) antibody and developed using an enhanced chemiluminesence (ECL) substrate reagent (Thermo Fisher Scientific).