Anti phospo p44 42 mapk erk1 2 clone e10

For detection of phosphorylation of Syk (pSyk) and ERK (pERK), 2.5 × 10⁴ immature moDCs were seeded overnight in a 96-well flat-bottom plate. moDCs were stimulated with SEA (50 μg/ml), SEAΔα-1/ω-1 (50 μg/mL), or ω-1 (500 ng/mL) in the presence or absence of blocking antibodies or inhibitors (R406, anti-MR, anti–Dectin-1, anti–Dectin-2, combination of anti–Dectin-1 and anti–Dectin-2 or IgG1 and IgG2 control antibodies) for indicated periods, and the moDCs were fixed for 15 min with 4% ultrapure formaldehyde (Polysciences, Warrington, PA) directly in the plate. The cells were harvested and washed first with PBS and then with 0.5% of saponin for permeabilization. Cell were intracellularly stained with anti–phospo-Try525/526 Syk (clone C87C1) and anti–phospo-p44/42 MAPK (Erk1/2) (clone E10) (both Cell Signalling Technology). Following 2-h incubation at room temperature, cells were washed with 0.5% of saponin, and Syk and ERK phosphorylation was determined by flow cytometry.

For the detection of ERK phosphorylation (pERK), 2.5 × 10⁴ immature moDCs were seeded overnight in a 96-well flat-bottom plate. moDCs were stimulated with SEA (25 μg/mL) and Δω1-SEA (25 μg/mL) for the indicated periods, and the moDCs were fixed for 15 min with 4% ultrapure formaldehyde (Polysciences, Warrington, PA, USA) directly in the plate. The cells were harvested and washed first with PBS and then with 0.5% of saponin for permeabilization. The cells were intracellularly stained with anti-phospo-p44/42 MAPK (Erk1/2) (clone E10) (both Cell Signalling Technology). Following 2 h of incubation at room temperature, the cells were washed with 0.5% of saponin, and ERK phosphorylation was determined by flow cytometry.