Simplestep elisa kit

The amount of total and phosphorylated ERK1/2 was quantified using SimpleStep ELISA Kit (Abcam, Cambridge, UK). Neutrophils (1 × 10⁶ in 500 µL of PBS per well) were stimulated with 100 µM and 200 µM FOH in 12-well microplate. Negative and positive controls were prepared as described above. After 1 h of incubation at 37 °C in 5% CO₂, cells were lysed using a Cell Extraction Buffer (Abcam, Cambridge, UK). The protein concentration in the lysate was determined using the Bradford assay [31 (link)]. Then, 50 µL of lysate was mixed with 50 µL of antibody cocktail (anti-ERK1/2–total or anti-pT202/Y204–phosphorylated ERK1/2 provided by the manufacturer) in wells of SimpleStep pre-coated 96-well microplate, according to manufacturer’s instruction. After 1 h of incubation with gentle shaking at room temperature, the wells were washed 3 times with PBS and then TMB (3,3′,5,5′-tetramethylbenzidine) solution was added. After 15 min, the reaction was stopped with a Stop solution, and absorbance at 450 nm was recorded using a BioTek Synergy H1 microplate reader.

Twenty-four hours after transfection, cells were serum-starved for 3 hours to reduce and stabilize basal levels of phospho-ERK1/2. Concentration response curves were generated by 1/5 serial dilutions as above. Drug solutions were prepared in serum-free DMEM. Cells were stimulated for 5 min with the different compounds and lysed immediately with ice-cold extraction buffer supplied in the ERK1/2 (pT202/Y204) SimpleStep ELISA Kit (Abcam ab176640; Cambridge, UK), according to manufacturer instructions. Total protein was also quantified by Bradford to normalize p-ERK1/2 response. Basal absorbance levels (vehicle treated cells) were subtracted form test samples and data expressed as percentage of 10 µM αMSH effect, normalized to 100%.

Western blotting and band quantification were performed as previously described^{24 (link),25 (link)}. Phospho-v-akt murine thymoma viral oncogene homolog (AKT) (S473) and total AKT antibodies (9271 and 9272) were from Cell Signaling (Danvers, MA USA) and actin antibody (A2066) from Sigma. Activation of Nuclear factor κB (NFκB) was assessed by quantification of subunit p65 phosphorylation (pS536) using the NFκB p65 (pS536) SimpleStep ELISA kit (ab176647, Abcam, Cambridge, UK). Absorbance values were normalized to total protein content measured with the Pierce BCA protein assay kit (Thermo Fisher Scientific, Lafayette, CO, USA).