Supersep ace 15 precast gel

Proteins were harvested from the intracellular and culture supernatants. Before collection, MH7A cells, FLS, monocytes, and macrophages were washed with phosphate-buffered saline. The cells were dissolved in radioimmunoprecipitation assay (RIPA) buffer. For the supernatant protein assay, the last 24 h of incubation was performed in a serum-free medium. Cell supernatants for FLS, MH7A, HEK293T, and macrophages were suspended in acetone and kept at –20 °C for 1 h. The samples were then centrifuged for 10 min at 15,000 × g at 20–25°C. Protein precipitates were dried for 30 min and dissolved in RIPA buffer. Proteins were processed using a SuperSep Ace 15 % precast gel (FUJIFILM Wako Pure Chemical Co.) and transferred onto a polyvinylidene fluoride membrane. The membranes were probed with anti-human β-actin antibody (0.1 μg/mL, mouse monoclonal, clone AC-15, Sigma-Aldrich), anti-human FXIII-A antibody (0.025 μg/mL, mouse monoclonal, Proteintech), anti-human FXIII-B antibody (0.1 μg/mL, rabbit polyclonal, Sigma-Aldrich), and anti-HA-tag antibody (0.2 μg/mL, goat polyclonal, Genscript). Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) were then added. Horseradish peroxidase activity was detected using ECL prime reagents (Cytiva, Tokyo, Japan), followed by imaging using the Image Quant LAS 500 (Cytiva) system.