Anti caspase 1

For short-term cultures, gingival fibroblasts were serum-starved for 24 h, then treated with 1 μg/ml LPS for 24 h. For long-term cultures, gingival fibroblasts were treated with 1 μg/ml LPS in α-MEM containing 2% FBS for 14 days. Proteins were detected following a previously published western blot procedure [15 (link)]. The following proteins were detected by the following antibodies: anti-caspase 1 (Cat# 14367; Gene Tex Inc., Irvine, CA), anti-caspase 3 (Cat# 611048; BD Transduction Laboratories, San Jose, CA), anti-IL-1β (Cat# AF-201-NA; R&D Systems Inc., Minneapolis, MN), anti-NLRP3 (Cat#LS-C162911; LifeSpan Biosciences, Inc., Seattle, WA), anti-NF-κB p65 (Cat#3033; phosphorylated Ser 536, Cell Signaling Technology, Inc., MA, USA), anti-NF-κB p65 (Cat# sc-71675; Santa Cruz Biotechnology, Inc., Dallas TX), anti-IKKβ (Cat# ab32041; Abcam, MA), anti-IKK beta (Cat# ab59195; phosphorylated Y199, Abcam), β-actin (Cat# sc-69879; Santa Cruz Biotechnology, Inc.).

Cells were lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 50 mM Tris-HCl [pH 8.0]) or SDS buffer (2% SDS, 50 mM Tris-HCl [pH 7.5]) containing protease inhibitor and phosphatase inhibitor cocktail (Roche). Harvested extracts were separated by 10% SDS-PAGE and transferred to PVDF membranes. Later, the membranes were incubated with primary antibody, and then with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratory, West Grove, PA) and were finally visualized using enhanced chemiluminescence (Thermo Fisher Scientific). Image acquisition and signal density measurements were carried out using the BioSpectrum Image System (UVP, Upland, CA). The following primary antibodies were used: anti-caspase 1 (GTX111630, GeneTex, Irvine, CA), anti-caspase 3 (#9665, Cell Signaling Technology, Danvers, MA), anti-caspase 7 (GTX1002337, GeneTex), anti-caspase 8 (#4790, Cell Signaling Technology), anti-bone morphogenetic protein 4 (BMP-4; GTX100875, GeneTex), anti-phospho-STAT1 (phospho-Tyr701, #9167 Cell signaling), anti-STAT1 (#14994, Cell Signaling), anti-phospho-STAT2 (phospho-Tyr690, GTX50721, GeneTex), anti-STAT2 (#14994, Cell Signaling), anti-DENV NS3 (GTX124252, GeneTex), and anti-GAPDH (#60004-1-Ig, Proteintech Group, Rosemont, IL).