Anti caspase 4

THP-1-derived macrophages were serum-starved for 24 h followed by treatment with 1 μg/mL P. gingivalis LPS and/or 10 μM caspase-1/-4/-5 inhibitor Ac-FLTD-CMK at 5% CO₂ for 6 h. Cells were collected, and proteins were extracted using a Total Protein Extraction Kit (Signalway Antibody, Greenbelt, MD, USA) according to the manufacturer’s instructions. Equal amounts of protein were separated using 10% SDS–PAGE (Bio–Rad) and transferred to polyvinylidene difluoride membranes (GE Healthcare Life Science, Pittsburgh, PA, USA). The membrane was incubated in a blocking buffer (5% BSA in Tris-buffered saline containing 0.1% Tween 20) at room temperature for 1 h and then incubated with the following primary antibodies overnight at 4 °C: anti-caspase-1 (sc-398715, Santa Cruz Biotechnology), anti-caspase-4 (sc-56056, Santa Cruz Biotechnology), anti-caspase-5 (sc-393346, Santa Cruz Biotechnology), anti-GADMD-N (ab215203, Abcam), anti-IL-1β (ab9722, Abcam) and anti-GAPDH (Signalway Antibody). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at 37 °C. A chemiluminescence kit (Bio-Rad, Hercules, CA, USA) was used to visualize the immunoreactive bands. The bands were analyzed by ImageJ software.