In vitro kinase assays

Six micrograms (6 μg) of purified MCU NTD_WT, 20 μg of purified MCU NTD^AA_S92, and 32 μg of commercially obtained MBP (Enzo, ALX-202-075) were phosphorylated by PKA (Promega, V5161), PKC mixtures (α, β, and γ isoforms with lesser δ and ζ; Promega, V5261), PKC_βII (Promega, V3741), PKC_δ (Promega, V3401), and PKC_ε (Promega, V4036). In vitro phosphorylation of PKA was performed in 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 5 mM β-glycerophosphate, 0.1 mM Na₃VO₄, 0.2 mM Mg-ATP, and 3 pmol of [γ-³²P]ATP (3000 Ci/mmol) with 20 ng PKA for MBP and 100 ng PKA for MCU NTD_WT and MCU NTD^AA_S92. In vitro phosphorylation assays of PKC mixtures, PKC_βII, PKC_δ, and PKC_ε were performed in 1 × reaction buffer A (SignalChem, K03-09) [20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 0.02% (v/v) Tween-20], 2 mM DTT (SignalChem, D86-09B), 1 × PKC lipid activator (SignalChem, L51-39), 0.2 mM Mg-ATP, and 3 pmol of [γ-³²P]ATP (3000 Ci/mmol) with 100 ng PKC mixtures, PKC_βII, PKC_δ, and PKC_ε for MBP and 100 ng PKC, 50 ng PKC_βII, 200 ng PKC_δ, and 200 ng PKC_ε for MCU NTD_WT, and MCU NTD^AA_S92. All in vitro kinase assays were performed at 30 °C for 60 min. The reaction was halted by the addition of SDS-PAGE sample buffer. Then, reaction samples were resolved by SDS-PAGE and visualized by autoradiography.