Anti p syk

Cell culture and DNA transfection reagents were purchased from Invitrogen, Inc. (Carlsbad, CA). Recombinant murine RANKL and MCSF were obtained from R&D Systems, Inc. (Minneapolis, MN). Anti-NFATc1, anti-PLCɣ, anti-p-Syk, anti-Syk, anti-calcineurin and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-NFAM1 antibody was purchased from Bioss Antibodies Inc (Woburn, MA). Secondary antibodies conjugated to fluorophores (AlexaFluor-488 and AlexaFluor-568) were obtained from Invitrogen, Inc., and DRAQ5 was from Axxora Platform, San Diego, CA (Biostatus Ltd.'s distributors). SuperSignal enhanced chemiluminescence reagent was obtained from Amersham Bioscience (Piscataway, NJ), and PVDF membranes were purchased from Millipore (Bedford, MA). Fura-2, AM and probenecid were purchased from Life Technologies, Carlsbad, CA. Histochemical kit for tartrate-resistant acid phosphatase (TRAP) activity, Ficoll-Paque, and DNAse I was purchased from Sigma (St Louis, MO).

Replicate wells of THP-1 derived macrophages (4 x 10⁶ cells/well) were incubated as described with Cdt. The cells were washed and treated with 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate and protease and phosphatase inhibitors (ThermoFisher Pierce Protein Biology, Pittsburgh, PA); replicate wells were pooled and protein concentration determined. HMGB1 release into culture supernatants was determined following precipitation of supernatants in 20% TCA. Samples were separated on 12% SDS-PAGE and then transferred to nitrocellulose. The membrane was blocked with BLOTTO and then incubated with anti-pro-IL-1β, anti-caspase-4, anti-Syk, anti-pSyk, anti-GSDMD or anti-GAPDH antibody (Santa Cruz Biotechnology) for 18 hr at 4°C (Shenker et al., 1999 (link)). Membranes were washed, incubated with either goat anti-rabbit IgG or goat-anti-mouse IgG conjugated to horseradish peroxidase. The Western blots were developed using chemiluminescence and analyzed (Licor).