Anti phosphotyrosine antibody

LPS, isoamyl alcohol, 2-methylbutane, picrotoxin, 3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid, sodium orthovanadate, and monoclonal anti-mouse β-actin antibodies were all purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse ASC and caspase-1 antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX), anti-phosphotyrosine antibodies from Cell Signaling (Danvers, MA), anti-mouse and human IL-1β antibodies from R&D Systems (Minneapolis, MN), and anti-human caspase-1 antibodies from BIOMOL International LP (Plymouth Meeting, PA). Recombinant mature mouse IL-1β was also purchased from R&D Systems. Biotin conjugated anti- mouse and rabbit secondary antibodies were from GE Healthcare UK Limited (Little Chalfont, Buckinghamshire, UK) and anti-goat IgG from Jackson ImmunoResearch (West Grove, PA). For inflammasome stimulation, ATP, nigericin, alum Imject, apoSAA, poly (dA:dT), and anthrax lethal factor and protective antigen were purchased from Amersham Biosciences (Piscatawy, NJ), Invivogen (San Diego, CA), Thermo Scientific (Waltham, MA), PeproTech Inc. (Rocky Hill, NJ), Invivogen, and BEI Resources (Manassas, VA), respectively. Muscimol was acquired from MP Biomedicals (Santa Ana, CA), and ethanol from Pharmco AAPER (Brookfield, CT). ASC (Tyr144) phospho-specific antibody was purchased from ECM Biosciences (Versailles, KY).

Cell extract, preparation and immunoblotting were performed as previously described.^{2 (link),51 (link)} PYK2, STAT3 and JAK1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AKT and pSTAT3 (pY 705), and anti-phosphotyrosine antibodies were from Cell Signaling (Boston, MA, USA). Anti-pJAK1 (pYpY 1022/1023) was from Invitrogen (Grand Island, NY, USA). pERK1/2 (pT 202/pY 204), ERK1 and β₁ integrin antisera was from BD Biosciences (San Jose, CA, USA). pAKT (pS 473) antibody was from Imgenex (San Diego, CA, USA). Anti-pPYK2 (pY402) was from R&D Systems (Minneapolis, MN, USA). Actin antibody was from Sigma and horseradish peroxidase-conjugated secondary antibody was from Jackson ImmunoResearch (West Grove, PA, USA). STAT3 phosphorylation in primary cells was examined by FC. Cells were fixed in 1.6% paraformaldehyde, then permeabilized in 95% methanol for 10 min, and incubated in 1% fetal bovine serum overnight prior to staining with Alexa647-conjugated anti-pSTAT3 (pY 705; BD Pharmingen) for 2 h at 4 °C. Fibronectin was purchased from Invitrogen.