Alphalisa surefire ultra p erk 1 2 thr202 tyr204 assay kit

ERK phosphorylation assays were performed on HEK Flp-in hCB₂ and HEK Flp-in wt cell lines. Cells were seeded at a density of 4.5 × 10⁴ cells per well in poly-D-lysine treated clear 96-well Nunc^TM plates; for PTX experiments cells were plated in the presence of PTX (100 ng mL^-1) or vehicle. 24–25 h later, medium was removed and replaced with basal medium (DMEM supplemented with 1 mg mL^-1 BSA). Cells were equilibrated for 3 h at 37°C followed by drug stimulations carried out at 37°C for 4 min. All drugs were prepared in basal medium at 2× final concentration. An inhibitor of ERK phosphorylation, U0126 (Cell Signaling Technology, MA, United States) at 10 μM, and a pERK pathway stimulant, phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) at 100 nM, both incubated for 15 min, were utilised as reference points. At the conclusion of the drug incubation, plates were placed on ice, and immediately lysed by adding 20 μL of ice-cold lysis buffer (from AlphaLISA^® Kit; details follow). Detection was performed using the AlphaLISA^® SureFire^® Ultra^TM p-ERK 1/2 (Thr202/Tyr204) Assay Kit (PerkinElmer), according to manufacturer instructions, and plates read in a CLARIOstar^® reader (BMG Labtech) using standard AlphaScreen-compatible filters. Data were normalised to matched U0126 (0%) and PMA (100%) treatments, allowing compilation of data from independent experiments.

Quantification of cAMP in BMSC-derived neural cells after stimulation was carried out using a commercial kit (LANCE^® Ultra cAMP Kit). After the strain and/or electrical stimulation, the differentiated cells were collected and seeding at 1,000 cells per well in a white OptiPlate^TM-384 microplate and then followed the manufacturer’s guidance. The time-resolved fluorescence resonance energy transfer (TR-FRET) signal was measured on an EnVision^® Multilabel reader (PerkinElmer, United States). The cAMP level was calculated according to the standard curve.
The phosphorylation of ERK and AKT was detected by AlphaLISA^® SureFire^® Ultra^TM p-ERK 1/2 (Thr202/Tyr204) assay kit and AlphaLISA SureFire Ultra p-AKT1/2/3 (Thr308) Assay Kit, respectively (PerkinElmer, United States).