Phospho p44 42 mapk erk1 2 thr202 tyr204 antibody

Twelve-day-old seedlings (n=10) grown on liquid MS medium in 24-well plates were treated with water (mock) and oligosaccharides for 0, 10, 20, and 30 min, and then harvested in liquid nitrogen. Protein extraction and detection of activated MAPKs using the Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell Signaling Technology) were performed as described (Ranf et al., 2011 (link)). Western-blot shown is from one experiment representative of three independent ones with similar results.

After 48 h of transfection, the cells were stimulated with bFGF. Then, the total proteins were extracted using RIPA lysis buffer, and the protein concentrations were determined with a Bradford Protein Assay kit (Beyotime, China). Thereafter, equal amounts of protein were electrophoresed on SDS-PAGE gels and transferred to PVDF membranes, followed by blocking with 5% BSA for 1 h. Then, the membranes were incubated with an FGFR1 antibody (1:800, Proteintech, USA), GAPDH antibody (1:10000, Sungene Biotech, China), ERK1/2 antibody (1:1000, Bioworld, USA) or phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody (1:1000, Cell Signaling Technology, USA) at 4 °C overnight. The next day, after washing with TBST, the membranes were incubated with goat anti-rabbit IgG/HRP (1: 20000, Sungene Biotech, China) and goat anti-mouse IgG/HRP (1: 10000, Sungene Biotech, China) at 37 °C for 1 h. Finally, the signal was detected using ECL reagents (Beyotime, China).

HEK293 cells were cultured in Eagle’s Minimal Essential Medium (MEM) containing 10% FBS and 1% penicillin/streptomycin (P/S) and adjusted to 1.0 × 10⁵ cells/mL. Five milliliters of this solution were dispensed in T25 flasks and incubated at 37°C for 24 h. The following day, 5 μg of pcDNA3.1 (+), the expression vector of WT, p.Gly23Val, or p.Gly24Glu, were transfected into the HEK293 cells using Lipofectamine^® 3000 Reagent (Thermo Fisher Scientific, Waltham, MA). Next, the cells were cultured for 48 h and the protein was extracted using RIPA buffer. Western blotting was performed according to the standard protocol using Anti–RRAS2 Polyclonal Antibody (#PA5-22123, Invitrogen), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody (#9101, Cell Signaling Technology, Danvers, MA, United States)), p44/42 MAPK (Erk1/2) Antibody (#9102, Cell Signaling Technology, Danvers, MA, United States)) and Anti–GAPDH (14C10) Rabbit mAb (#2118, Cell Signaling Technology, Danvers, MA, United States) as the primary antibodies. Anti–rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling Technology) was used as the secondary antibody.

Hemocytes (2.5 × 10⁴ cells/μL) were stimulated with DA (10 μM) for 5, 15, or 30 minutes at 25 °C. In some experiments DOP1 receptor signaling was blocked by preincubation with the antagonist, SCH 23390 (Sigma; S7389) at the concentration of 1 nM and 10 μM for 60 minutes at 25 °C. Cell lysates were prepared, and proteins were resolved by SDS-PAGE. Blots were immunolabeled overnight at 4 °C using 1 μg/mL polyclonal rabbit Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody (Cell Signaling Technology, Beverly, MA) diluted in TBS-T. Secondary labeling, detection, and analysis were performed as previously described. Membranes were stripped and reprobed with polyclonal rabbit p44/42 MAPK (ERK1/2) antibody (1 μg/mL; Cell Signaling Technology).

To perform Western blot assays, three-week-old rosettes of A. thaliana grown in MS1/2 medium were treated with 100 nM fls22 or infested with 20 adult female mites for 1h, 3h, and 24h. Eight rosettes per genotype were sampled and harvested in liquid nitrogen. MAPK activation was detected by immunoblot analysis of soluble proteins extracted from seedlings in a lysis buffer, using the Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell Signalling Technology).

Ten μg of each protein extract were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane (HybondC, Amersham, United Kingdom) by semi-dry electroblotting transfer (Trans-Blot SD, Bio-Rad, United States) for 40 min at 15V, using transfer solution [48 mM Tris; 39 mM glycine; 0.0375% SDS (m/v), 20% methanol (v/v)]. A membrane-blocking step was performed for 1 h in TBS-T buffer [10 mM Tris–HCl pH 7.5; 150 mM NaCl; 0.05% Tween 20 (v/v)] supplemented with 2% BSA (m/v) on a rotary shaker before being incubated 1 h at room temperature, with primary polyclonal phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204) antibody (1/5000, Cell Signaling Technology, United States) or primary polyclonal ERK1/2 (total) antibody (1/1000, ab196883 Abcam plc., United Kingdom). Then, three washes were performed with TBS-T for 10 min, and the membranes were incubated 1 h at room temperature with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1/10000, Bio-Rad, United States). Immunoreactive proteins were revealed by electro-chemiluminescence (ECL, LumiGLO, Cell Signaling Technology, United States).