Rabbit anti erk1 2 9102

Phosphorylation of p44/42 mitogen-activated protein kinases (ERK1/2) was determined based on activation-associated phosphorylation. Cells cultured in six-well plates were serum starved for 4 hr before stimulation. Samples were treated with the indicated compounds at 50 μM or 0.5% DMSO (vehicle) for 10 min and then stimulated with fMLF (20 nM) for various time periods (0 – 15 min) or 200 ng/ml of PMA. The reactions were terminated by adding 150 μl of ice-cold SDS-PAGE loading buffer. Samples were analyzed by SDS-PAGE and Western blotting using rabbit anti-ERK1/2 (9102) and rabbit anti-ERK1/2 phosphorylated at Thr202 and Tyr204 (9101) (1:1000; Cell Signaling Technology, Inc., Danvers, MA). Horseradish peroxidase-conjugated AffiniPure goat anti-rabbit IgG (1:3000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used as secondary antibody. The immunoblots were visualized using a SuperSignal West Pico Chemiluminescence kit (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. The results of Western blotting were quantified using ImageJ software from the National Institutes of Health.