Stat3 py705 total elisa kit

All cytokine measurements were obtained from cell culture supernatants using Biolegend ELISA Max Plates or a Human Inflammatory Cytokine Multiplex Bead Assay of up to 27 cytokines (Bio-Rad) as per manufacturers’ instructions. A full list of cytokines measured by Human Inflammatory Cytokine Multiplex Bead Assay can be found in the Table 1. STAT3 and Phosphorylated STAT3 were measured using STAT3 (pY705 + Total) ELISA Kit (Abcam). Supernatants and cell lysates collected from each condition were stored at -80C prior to analysis. For each measurement, the supernatants were thawed, centrifuged to remove debris, and diluted in the provided buffer to concentrations necessary to fall within the reference range of the ELISA standard curve.Table 1

Measured Cytokines.

FGF basic	IFN-γ	IL-4	IL-8	IL-13	MCP-1	RANTES
Eotaxin	IL-1β	IL-5	IL-9	IL-15	MIP-1a	TNF-a
G-CSF	IL-1ra	IL-6	IL-10	IL-17	MIP-1b	VEGF
GM-CSF	IL-2	IL-7	IL-12 (p70)	IP-10	PDGF-BB

Semiquantitative measurements of STAT3 phosphorylated at Tyr705 and total STAT3 proteins in cell lysates were performed using the STAT3 (pY705) + Total ELISA Kit (Abcam, Cambridge, CB2 0AX, UK) according to the manufacturer’s instructions. Briefly, 1 × 10⁵ cells/well were seeded in a 6-well plate and treated with the test compounds for 24 h. The cells were solubilized using a chilled 1× cell extraction buffer PTR, and the sample protein concentrations were determined using a BCA protein assay (Thermo Fisher, Waltham, MA, USA). Samples were diluted to 300 ng/μL in a 1x cell extraction buffer PTR, and 50 μL of all samples and controls were added to the appropriate wells. Then, 50 μL of the antibody cocktail was added to each well. The plate was sealed and shaken at 400 rpm for 1 h at room temperature. The plate was further washed three times with a wash buffer. After removing any excess liquid, 100 μL of a TMB substrate were added to each well, after which the samples were incubated for 15 min in the dark while being shaken at 400 rpm. The plate was read at an OD of 450 nm after adding 100 μL of a stop solution to each well.