Anti asc ab

Huaier aqueous extract was obtained and prepared as described [24 (link)]. DSS (5000 kDA) was from Wako Pure Chemical Industries(Osaka, Japan); ATP, chloroquine, PMA and LPS (Escherichia coli, 055:B5) were from Sigma-Aldrich(St. Louis, MO, USA); MG132 was from Merck-Millipore(Temecula, CA, USA); 3-MA was from Selleck Chemicals(Houston, TX, USA) ; cycloheximide(CHX) was from ApexBio(Houston, TX, USA); anti-NLRP3 Ab and anti-ASC Ab, anti-caspase-1 p10 Ab and p20 Ab were from Adipogen(San Diego, CA, USA); anti-LC3B Ab was from Cell Signaling Technology(Trask Lane Danvers, MA, USA); anti-IL-1β was from Novus Biologicals(Littleton, CO, USA).

THP-1 cells were plated at 10⁵ cells/well in 96-well-plates in 100 μl IMDM 10% FBS without/with 50 nM PMA (phorbol 12-myristate 13-acetate, Sigma) and incubated at 37 °C for 18 h. Cells were then incubated for additional 24 h with the following stimuli: 1 μg/ml LPS and 96 h supernatant from PDAC cell lines (100 μl). In inhibition experiments, anti-IL-1α (10 μg/ml), anti-TNF-α (10 μg/ml), anti-IL-18 (10 μg/ml) and BoxA (10 ng/ml) or iso-Ig Abs (10 μg/ml) were added directly in the culture or THP1 cells were stimulated with ASC- or irrelevant iso-Ig Ab-depleted supernatants (100 μl). Depleted supernatants were obtained as follows: cell-free BxPC-3 and A8184 supernatants (from 72 h culture in IMDM 10% FBS) were incubated for 2 h at RT in 96-well-plates (not-treated sterile Costar #3788) coated with 1 μg/ml anti-ASC Ab (AL177 Adipogen) or normal rabbit control IgG (Peprotech), supernatants were then collected and stored at − 20 °C. THP1 were also stimulated with the supernatant of tumor cells that had been previously treated with either ASC siRNA or a negative control siRNA. IL-1β in the supernatants of THP1 cells was measured by ELISA.