Anti rabbit hrp secondary antibodies

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).

Thymocytes were lysed as previously described (Huang et al., 2007 (link)) in 1% Triton X-100/60 mM octylglucoside/150 mM NaCl/25 mM Tris-HCl, pH 7.5/1 mM EDTA containing Roche Complete Mini Protease Inhibitor and PhosSTOP Phosphatase Inhibitor Cocktails. Lysates were incubated for 20 min at 4°C, then cleared by centrifugation at 14,000 g for 10 min at 4°C. For immunoprecipitations, pre-cleared lysates were incubated for 1.5 hr with anti-Itpkb antibodies (G-20, Santa-Cruz Biotechnology) followed by incubation with Protein G-conjugated beads for 1.5 hr. Beads were washed 3 times with 1x lysis buffer, denaturated in 1x sample buffer at 99°C for 10 min and analyzed via SDS-PAGE/immunoblot. For immunoblot analysis, nitrocellulose membranes were incubated overnight at 4°C with anti-Itpkb (#AP8167b, Abgent) or anti-PLCγ1 (#2822, Cell Signaling Technology) antibodies and then for 45 min with anti-rabbit-HRP secondary antibodies (Bio-Rad Laboratories) in TBS. Bound antibodies were detected by enhanced chemiluminescence (ECL kit, GE Healthcare).