Phospho tracer elisa kits

A Superdex 200 (GE Healthcare) gel filtration column was first equilibrated with 1.5 CV of endotoxin free PBS (Hyclone) at room temperature. Sestrin2 complexes were injected at time 0 (500 ul) and eluted using a single isocratic wash, fractions collected, and analyzed by ELISA-based binding assays (absorbance at 450 nm) across the spectrum for the indicated proteins. Phosphorylated MAPKs were detected using Phospho-Tracer ELISA kits (Abcam) as above described. For detection of Mios (MBS9330396); RagA (MBS9334409); and RagB (MBS9318748). The column was calibrated with protein markers as described 18 (link)

Sestrin or AMPK Immunoprecipitates were washed twice in lysis buffer and twice in kinase buffer (all from Cell Signaling). Kinase reactions were incubated for 30 min at 30 °C either in the absence or in the presence of 200 μM ATP (Cell Signaling), as indicated. MAPK activity was assessed using Phospho-Tracer ELISA Kits according to the manufacturer’s instructions (Abcam; see below). In some experiments, exogenous recombinant human Sestrin proteins (Genway; 1 μg/mL), the AMPK agonist A-769662 (150 μM), the Erk inhibitor FR18024 (20 μM); the Jnk inhibitor SP-600125, (10 μM); or the p38 inhibitor SB-203580, (10 μM) were added directly to the in vitro kinase reaction itself for 30’, as indicated. In vitro kinase assays are shown as proportional to fold increase at 450 nm absorbance emission of triplicate wells ± s.e.m, normalized to the total amounts of co-immunoprecipitated MAPKs.