Anti perk1 2 antibodies

Immunoprecipitation of COX isozymes and immunoblot analysis were performed as described using isozyme specific antisera48 (link)49 (link). For immunodetection of ERK and AKT proteins, frozen pancreas powder was homogenized in RIPA buffer (150 mM NaCl, 20 mM Tris/HCl pH 8.0, 1% Triton X-100, 1% C24H39NaO4, 1 mM PMSF, 2 mM EDTA, 0.1% SDS, 25 mM NaF, 1 mM Na₃VO₄, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.2 mg/ml α₂-macroglobulin). Immunoblots were incubated with anti-AKT, anti-p-AKT, anti-ERK1,2, anti-p-ERK1,2 antibodies (Cell Signaling, 1:1000 dilution) or anti-Notch1 antibodies (Cell Signaling; 1:2000) overnight at 4 °C. Peroxidase-coupled secondary antibodies (1:8000- 1:10000 diluted) were incubated at RT for 1 h. For detection of signals, the ECL western blotting detection kit was used (Amersham BioSciences, Freiburg/Germany). For Notch1 blots, the TE671 cell lysate served as positive control (Santa Cruz Biotechnology, Heidelberg/Germany, SC-2416). Cell monolayers transfected for 24 and 48 hours with siPtgs2 or siAllstars negative (n = 3, each) were washed twice with PBS and frozen at −20 °C prior to extraction with HP-buffer. The extracted protein served, after precipitation with ethanol and denaturation in Laemmli sample buffer (LSB), as a loading control. The cell monolayers were then reextracted with 2x LSB and denatured for immunodetection of Notch1 and COX-2.

Cultured cells were harvested and lysed on ice with lysis buffer, containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 0.5% Triton X-100, 10 mM NaF, 1 mM sodium orthovanadate, 10% glycerol and protease inhibitors (Calbiochem, San Diego, USA), for western blot. Protein concentration was determined by Bradford assay (Bio-Rad, Hercules, USA). Proteins were resolved by 10 % SDS-PAGE gels and transferred to nitrocellulose membranes. Visualization of the proteins was performed by chemiluminescence (AGFA, CP1000, Mortsel, Belgium). The anti-LysRS antibody was purchased from Neomics (Seoul, Korea). The anti-phosphorylated-LysRS antibody was custom-made against s207 phosphorylation (Abmart Laboratories, China). The anti-EGFR antibody was purchased from Santa Cruz Biotechnology (Dallas, USA), the anti-GluProRS and GlyRS antibodies from Abcam (Cambridge, UK), anti-ERK1/2 and anti-pERK1/2 antibodies from Cell Signaling Technology (Danvers, USA).

Seven-day-old Arabidopsis seedlings grown on 1/2MS plates were transferred to sterile water in a 12-well plate and were kept overnight to avoid possible wounding responses caused during collection. The plants were treated with 100 nM flg22 for the indicated times. Total proteins of each sample were extracted from 12 seedlings in 80 μL of extraction buffer (50 mM Tris-HCl [pH 7.5], 5 mM EDTA, 150 mM NaCl, 1% [v/v] Triton X-100, 1 mM NaF, 1 mM Na₃VO₄, 1 mM DTT, 1× complete protease inhibitors [Roche]). The supernatant was collected by centrifugation at 13,000 × g for 5 min at 4 °C, mixed with 2× protein sample buffer, and subjected to immunoblotting analysis. MPK6/3/4 activation was detected with anti-pErk1/2 antibodies (1:2000, Cell Signaling Technology, Cat. # 9101). GAPDH was used as the loading control, which was detected by immunoblotting with anti-GAPDH antibodies (1:3000, Proteintech, USA, Cat. # 60004-1).