Phospho p44 42 mapk erk1 2 rabbit monoclonal antibody mab

The MAPK activity of crude protein extracts from the cotyledons of 8-day-old seedlings treated with 1 µM flg22 for 1 h was determined as previously described (Flury et al., 2013 (link)). Specifically, the proteins from the crude extracts were separated by 10% SDS-PAGE and then transferred to a PVDF membrane (Bio-Rad; www.bio-rad.com) using a Mini-Protean II semi-dry electroblotting system (Bio-Rad). Activated MAPKs were detected following a 1 h incubation with Phospho-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (mAb) (1:2,000; Cell Signaling Technology, www.cellsignal.com) and a subsequent 1 h incubation with anti-rabbit-HRP secondary antibodies (Bio-Rad). The signals were visualized using a Clarity™ Western ECL system (Bio-Rad).

MAPK activity was determined using crude protein extracts from 8-day-old seedlings treated with 1 µM flg22 for 15–60 min as previously described [82 (link)]. Crude extracts were separated using 12% SDS-PAGE, and proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, www.bio-rad.com) using semi-dry electroblotting (Mini-Protean II system; Bio-Rad). Activated MAPKs were detected following 1 h incubation with Phospho-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (mAb) (1:2000; Cell Signaling Technology, www.cellsignal.com), followed by subsequent 1 h incubation with anti-rabbit-HRP secondary antibody (Bio-Rad). The signals were visualized using an enhanced chemiluminescence system (Clarity Western ECL; Bio-Rad). Protein detection and immunoblot analyses were performed according to the manufacturer’s instructions using mouse anti-FLAG as the primary antibody (M2, diluted 1/500; F3165, Sigma–Aldrich, St Louis, MO, USA). The secondary antibody was labeled with anti-Mouse–HRP secondary antibody (Bio-Rad) and diluted to 1/1000.