Anti p44 42 erk antibody

In vitro phosphorylation assays were performed as previously described [57 (link)]. Recombinant glutathione S-transferase (GST)-OsMKK6 or GST-OsMKK6^DD (500 ng) was incubated with the substrate proteins maltose binding protein (MBP)-OsMPK4 or MBP-OsMPK4m (1 μg) in kinase reaction buffer (30 mM Tris-HCl pH 7.5, 1 mM EGTA, 10 mM MgCl₂, 1 mM DTT, and 3 μL 10 mM ATPγS [Abcam, ab138911]) at 30 °C for 30 min. After adding 1.5 μL 50 mM p-Nitrobenzyl mesylate (PNBM, Abcam, Cambridge, UK; ab138910) to the reactions, the samples were incubated at 30 °C for 1.5 h. Recombinant proteins were separated by 10% (w/v) SDS-PAGE, and phosphorylated recombinant MKK6, MKK6^DD, MPK4, and MPK4m were detected by immunoblotting with anti-thiophosphate ester rabbit monoclonal antibodies (Abcam, Cambridge, UK; ab92570, 1:5000).
To detect MAPK activation, WT and rsr25 plants were inoculated with Xoo, and total proteins were extracted from the plants at 0 h, 48 h, 96 h, and 120 h after inoculation. MPK activation was detected by immunoblotting with an anti-p44/42 ERK antibody (Cell Signaling, Danvers, MA, USA; 4370S).

Type 1 insulin-like growth factor receptor levels were analyzed by Western blotting performed as described in detail elsewhere (12 (link)) and using a rabbit anti-IGF-IR antiserum (C-20, Santa Cruz Biotechnology, Dallas, TX, United States) and a horseradish peroxidase-conjugated donkey anti rabbit IgG antibody (GE Healthcare Life Sciences, Pittsburgh, PA, United States). To normalize for loading, the membranes were stripped and re-probed with a monoclonal anti-tyrosine tubulin antibody (Sigma-Aldrich, St. Louis, MO, United States). To analyze ERK1 and 2 phosphorylation in response to IGF-I, tumor cells, cultured overnight in serum-free medium, were stimulated with 100 ng/ml IGF-I (US Biological, Salem, MA, United States) for 10 min, lysed on ice in the presence of phosphatase inhibitors and the cell lysates separated on 8.5% SDS-polyacrylamide gels. The blots were probed, first with an anti-phospho-p44/42 ERK (Thr202/Tyr204) antibody and then with an anti p44/42 ERK antibody (Cell Signaling, Whitby, ON, Canada).