Igg hrp conjugated secondary antibody

M-199, RPMI-1640, yeast peptone dextrose medium, Giemsa dye, Trypan blue, MTT, 2′,7′-dichlorodihydrofluorescein diacetate dye, N-acetyl l-cysteine (NAC), R-mevalonate lithium salt, phosphoenolpyruvate, lactate dehydrogenase, pyruvate kinase, β-NADH, ATP, glycine, diphenyleneiodonium chloride, ammonium bicarbonate, urea, sodium chloride, magnesium chloride, manganese chloride, zinc chloride, calcium chloride, copper sulphate, proteinase K, ergosterol (ERG), diphenyl hexatriene (DPH), RNase A, TRIZOL, nitro blue tetrazolium, the lactate dehydrogenase (LDH) assay kit, caprylic acid, 8-amino caprylic acid, 4-methyl octanoic acid, methyl octanoate, valproic acid, the Apoptosis Detection Kit, Taq Polymerase for PCR, Anti-His primary antibody and IgG-HRP conjugated secondary antibody and all solvents were from Sigma-Aldrich Co. (St Louis, MO, USA). The QIAamp DNA Mini kit, RNeasy Mini Kit, High fidelity Taq DNA Polymerase, and Ni-NTA agarose were from Qiagen. from Thermo-Fisher. The cDNA synthesis kit was from Hoffmann-La Roche (Basel, Switzerland). Trypsin, iodoacetamide, and DTT, restriction enzymes like BamHI, HindIII, and XhoI were purchased from Thermo Fischer (Waltham, MA, United States). The RAW 264.1 and THP-1 cell line was obtained commercially from the National Cell Repository, NCCS, Pune.

Blots of cell lysates were incubated with antibodies against the following proteins: Caspase-1p10 (M-20 Cat. Number sc-514, Santa Cruz Biotechnology), NLRP3 (Cat. Number ab4207, Abcam), NLRC4 (Cat. Number 06-1125, Millipore) and pNLRC4 (Genetech), LC3b-I or LC3b-II (Abcam) followed by IgG–HRP-conjugated secondary antibody (Sigma–Aldrich) after separation in 12% Tris/glycine SDS gel and transfer to a nitrocellulose membrane. Normalization was performed probing the membrane with mouse-anti-β-actin antibody (Sigma–Aldrich). Chemiluminescence detection was performed with LiteAblotPlus chemiluminescence substrate (Euroclone S.p.A), using the ChemiDocTM XRS+Imaging system (Bio-Rad), and quantification was obtained by densitometry image analysis using Image Lab 3.1.1 software (Bio-Rad). Images have been cropped for presentation. Full size images are presented in Supplementary Figs 12-14.

Cells were lysed in 2x Laemmli buffer (Sigma-Aldrich). Blots of cell lysates were incubated with an antibody against IDO1 (clone 10.1, Millipore) followed by IgG–HRP-conjugated secondary antibody (Sigma–Aldrich) after separation in 10 or 12% Tris/glycine SDS gel and transferred to a nitrocellulose membrane. A cell lysate of IFNγ-stimulated HeLa cells was used as positive control in selected experiments for correct assignment of IDO1 band. Normalization was performed probing the membrane with mouse-anti-β-tubulin antibody (Sigma–Aldrich, clone T9026). Chemiluminescence detection was performed with LiteAblotPlus chemiluminescence substrate (Euroclone S.p.A), using the ChemiDocTM XRS+Imaging system (Bio-Rad), and quantification was obtained by densitometry image analysis using Image Lab 6.0 software (Bio-Rad).