Mab1694

Protein extraction was mediated by RIPA lysis buffer (89901; Thermo Fisher Scientific) supplemented with phosphatase inhibitors (PhosSTOP; Roche) and protease inhibitors (cOmplete; Roche). After determining protein concentrations with Pierce BCA protein assay kit (Thermo Fisher Scientific), proteins were reduced using NuPAGE reducing agent (Thermo Fisher Scientific) and NuPAGE LDS sample buffer (Thermo Fisher Scientific) and loaded on 4%–15% acrylamide Mini-Protean TGX gel (Bio-Rad). After running gel electrophoreses, proteins were transferred to a PVDF membrane and blocked in 5% BSA in Tris-buffered saline with Tween (TBST). Primary antibody used was anti-FSTL1 (1:1,000; MAB1694; R&D Systems), and detection was mediated by secondary anti-rat horseradish peroxidase (HRP)-conjugated antibody (1:2,000; 31470; Thermo Fisher Scientific) in a Bio-Rad ChemiDoc Imager system.

100 μL serum samples were added to the wells of the assay plate, which was coated with 5 μg/mL human FSTL1 capture antibody (R&D system, AF1694). The plate was incubated at 4 °C overnight, then 2.5 μg/mL biotinylated human FSTL1 detection antibody (R&D system, MAB1694) was added followed by washing with wash buffer and the plate was incubated for 1 hour at room temperature. Streptavidin-HRP was added to the wells of assay plate and incubated for another 30 min at room temperature. After washing the plate again, TMB substrate was added to each well and incubated in the darkness for 15 min at room temperature. The absorbance values of the wells at 450 nm and 570 nm were obtained after the stop solution was added. The ELISA assay was performed in duplicate for every sample.