Z atad fmk

Fibrinogen, lipopolysaccharide from Escherichia coli (055:B5; LPS), tunicamycin, thapsigargin, leucine-methyl-ester (LME), staurosporine, hirudin, and Isolectin B₄ from Griffonia simplicifolia-FITC conjugated were from Sigma (Dorset, United Kingdom) and thalidomide was from Tocris Bioscience (Bristol, United Kingdom). Quantikine M TNFα. TGF-β and IL-6 ELISA kits, z-VAD-FMK and z-ATAD-FMK were from R&D Systems (Abingdon, United Kingdom). Caspase 12 (FITC-ATAD-FMK) activity kits were from Promokine (Heidelberg, Germany) and caspase 3/7 (FAM-DEVD-FMK) activity kits were from Millipore (Watford, United Kingdom). Anti-ED1, anti-CD11b and rat IgG isotype controls were from AbD Serotec (Kidlington, United Kingdom), anti-caspase 12 and goat anti-rat-FITC were from Abcam (Cambridge, United Kingdom). Goat anti-rabbit IgG horseradish peroxidase (HRP) and goat anti-mouse IgG HRP were from Autogen Bioclear (Calne, United Kingdom).

Chemicals and inhibitors were dissolved in dimethyl sulfoxide and diluted to the desired concentrations directly in the culture medium. RAW 264.7 cells were pretreated with the indicated concentrations of inhibitors for 1 h before M. smegmatis infection. TM, z-LEHD-fmk (caspase-9 inhibitor), z-VAD-fmk (pan-caspase inhibitor), SP600125 (p-JNK inhibitor), PD98059 (p-ERK inhibitor), SB203580 (p-p38 inhibitor), BAY 11-7082 (NF-κB inhibitor), and caffeic acid phenethyl ester (NF-κB inhibitor) were purchased from Calbiochem (San Diego, CA, USA). NAC, cytochalasin D, staurosporine, and 4-PBA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Z-ATAD-fmk (caspase-12 inhibitor) was purchased from R&D Systems (Minneapolis, MN, USA). Lipopolysaccharide and H₂O₂ were purchased from InvivoGen (San Diego, CA, USA).
Western blotting was performed using antibodies against caspase-12, caspase-9, caspase-3, PDI, ERO1α, p-protein kinase RNA-like ERK, p-eIF2α, CHOP, Bip, p-p38, p-JNK, p-ERK, p-IκBα, IκBα, JNK, ERK, p38 (Cell Signaling, Danvers, MA, USA), and ATF6α (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Goat anti-rabbit IgG (Cell Signaling) and goat anti-mouse IgG (Calbiochem) were used as secondary antibodies. β-actin (Santa Cruz Biotechnology) was used as a loading control.