Erk 1 2 total phospho instantone elisa kit

HT-29 cells were seeded onto a 96-well plate and cultured until 80% confluency. Different concentrations of polyP1150 (10, 50, 250, and 500 mM), were incubated at 37°C for 4 h with or without phytase (42.9 mU/mL). Aliquots (10 μL) of the reaction mixtures were added to the HT-29 cells at final concentrations of 1, 5, 25, and 50 mM polyP1150 at 37°C for 6 h and incubated. The cells were also exposed to 50 mM polyP1150 with or without wheat phytase at 37°C for 1, 6, 12, and 24 h in a CO₂ incubator. The activity of total and phospho p38 in the HT-29 cells was measured at OD 450 nm using the p38 (Total/Phospho) InstantOne ELISA kit (Thermo Fisher Scientific, USA), and the amount of total and phospho ERK 1/2 in the HT-29 cells was evaluated at OD 450 nm using the ERK 1/2 (Total/Phospho) InstantOne ELISA kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.

To assess phosphorylation levels of p38 MAPK and ERK1/2, cells were stimulated with exosomes for 0, 5, 10, 30 or 60 min, or with insulin as a positive control. Cells were washed with ice-cold PBS and rapidly lysed on ice using lysis buffer provided in the kit with protease and phosphatase inhibitors. p38 MAPK activation was assessed with p38 MAPK alpha (pT180/pY182) + total p38 MAPK alpha ELISA Kit (Abcam ab126453) per manufacturer’s instruction. ERK1/2 activation was measured with ERK 1/2 (Total/Phospho) InstantOne™ ELISA Kit (Thermo Fisher) per the manufacturer’s instruction. Absorbance was read at 450 nm on a microplate reader.