Flag tag antibody

Western blotting was performed as previously described [17 (link)]. Briefly, total protein was extracted using RIPA buffer (Beyotime Biotechnology), resolved on SDS-PAGE gels and transferred onto PVDF membranes. Following blocking with non fat dry milk in TBST, membranes were washed and incubated with primary antibodies for (anti-LRP5, #5731, CST; NUP37, ab220675, Abcam; anti-CTGF, #86641, CST; Flag-tag antibody and myc-tag antibody from Genscript) overnight at 4 °C. Membranes were then incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature and detected with chemiluminescence using Immobilon Chemiluminescent HRP Substrate (Millipore). Results are representative of at least three independent experiments.

Dextran sulfate sodium salt (DSS) was purchased from MP Biomedicals (CA, USA), adenosine triphosphate (ATP) was obtained from In vivoGen Biotechnology (CA, USA), Methyl sulfoxide (DMSO), lipopolysaccharide (LPS), and thioglycolate were purchased from Sigma-Aldrich (MO, USA). Carbobenzoxy-Leu-Leu-leucinal (MG132), chloroquine (CQ), and cycloheximide (CHX) were purchased from Selleck (TX, USA). Dithiothreitol (DTT), ethylene diamine tetraacetic acid (EDTA), and primers used in this study were synthesized by Sangon Biotech (Shanghai, China). Antibodies against NLRP3, Caspase-1, and ASC were purchased from Adipogen Life Sciences (CA, USA). Antibody specific to IL-1β was obtained from Cell Signaling Technology. GSDMD antibody was purchased from Abcam (Cambridge, UK). The Flag-tag antibody was obtained from Genscript (Nanjing, China). β-actin and HA-tag antibodies were purchased from Proteintech (Wuhan, China).