Anti mouse igg fc specific peroxidase

The nanobody-Fc constructs were tested against influenza antigen standards from the National Institute for Biological Standards and Control (NIBSC) as listed in Supplementary Table 3. Plates were coated overnight with 50 μL/well of 5 μg/mL influenza virus antigen standard diluted in PBS at 4°C. Serial two-fold dilutions ranging from 4 μg/mL to 1 ng/mL of purified R1a-B6-mIgG1, R1a-B6-mIgG2a, R1a-B6, and cAb1-mIgG2a were then added. Secondary antibody, anti-mouse IgG (Fc Specific)-Peroxidase (Sigma A0168) at a 1:2000 dilution was used to detect nanobody-Fc; while monoclonal anti-polyHistidine-Peroxidase clone HIS-1 (Sigma A7058) at a 1:1000 dilution was used to detect R1a-B6. TMB substrate (Thermo Scientific 34029) was added to the plate and the reaction was stopped with 0.5 M HCl or 0.5 M H₂SO4. Plates were read on a SpectraMax M5 ELISA plate reader using SoftMax Pro software. Absorbance at 450 nm was taken.

LPS and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Sigma. ELISA kits were purchased from BlueGene. The Pierce BCA Protein Assay kit and Detoxi-Gel™ Endotoxin Removing Gel were purchased from Thermo. TRIzol Reagent was purchased from Life Technologies. Ni-NTA His Bind resin was purchased from Novagen. PrimeScript^™ RT reagent Kit with gDNA Eraser and SYBR Premix Ex Taq were purchased from Takara. High-glucose DMEM was purchased from HyClone. FBS was purchased from Gibco. The following antibodies were used: anti-ATF3 and secondary antibodies (anti-rabbit IgG (whole molecule)-peroxidase produced in goat (Sigma; 1:8000), monoclonal GAPDH (Sigma; 1:5000) and secondary antibodies (anti-mouse IgG (Fc specific)-peroxidase produced in goat (Sigma; 1:14000).

Cells in primary culture (27 × 10⁴ cells/well in 6-well culture plates) were allowed to grow until nearly confluence and then treated with IL-1β (10 ng/ml) or IL-1β+CM for different times. Cells were lysed with buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 10 minutes at 10000 xg. Protein content was determined by the DC Bio-Rad Laboratories protein reagent (Bio-Rad, Madrid, Spain). Proteins (25 μg) in supernatants were separated by SDS/PAGE (12.5% gel) and transferred on to PVDF membranes. Membranes were blocked with 3% BSA and incubated with specific polyclonal antibodies against p53 (Novus Biologicals, Littleton, CO, USA), acetyl-p53 (ChemiconMillipore Iberica, Madrid, Spain), P-p38 (Promega Corp., Madison, WI, USA), p38, JNK1/2, P-JNK1/2, ERK1/2, P-ERK1/2 (Cell Signalling Technology Inc.) overnight at 4°C. Membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (Dako, Copenhagen, Denmark); for p53 we used the secondary antibody anti-mouse IgG (Fc-specific)-peroxidase (Sigma-Aldrich). Finally, membranes with the immunoreactive bands were visualized by ECL® (enhanced chemiluminescence; GE Healthcare, Barcelona, Spain) using an AutoChemi image analyser (UVPInc., Upland, CA, USA).