Secondary horseradish peroxidase conjugated antibodies

SDS-PAGE was carried out on tissue homogenates with each sample standardized to 1.5 μg DNA per well. Proteins were separated on 4%–12% Bis-Tris Plus SDS gels and transferred to 0.45 μm polyvinylidene difluoride membranes (Life Technologies, Carlsbad, USA). Membranes were incubated overnight at 4°C with primary antibodies against carbonic anhydrase IX (CA-IX) (1/200, R&D Systems, AF2188), glucose transporter 1 (GLUT1) (1/1,000; Abcam, ab32551), phosphorylated histone protein γH2AX (1/2000; Abcam, ab11174), or ß-actin (1/10,000, Sigma, A5316), and for 1 h at room temperature with secondary horseradish peroxidase-conjugated antibodies (1/5,000, DAKO). β-Actin was used as loading control. For GLUT1, the same positive control (20 µg protein of 1% O₂ treated T24 cell lysate) was run on each gel to normalize signals between the blots. For CA-IX, a clear cell renal cell carcinoma tissue sample (University of Otago Human Ethics committee H14/020 (37 (link)), adjusted to 1.5 μg DNA per well, was similarly run on each gel. For γH2AX, a positive control (WiDr cells from ATCC treated for 4 h with 20 mM ascorbate, 20 µg protein) was run on each gel. Protein bands were visualized using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, USA), and quantified with the Alliance 4.7 imaging system and the ImageJ software (36 (link), 37 (link)).

Levels of endogenous AAT were estimated in plasma and PL fluid samples obtained at 20 h post-surgery from untreated septic and sham mice by Western blotting. In brief, 1 µL of plasma samples and 5 µL of PL fluid samples were separated on 10% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE). From gels, proteins were transferred onto polyvinylidene fluoride membrane by semidry Western blotting. For specific detection of AAT, primary rabbit polyclonal anti-AAT antibody was used at a dilution of 1:800 (DAKO, Glostrup, Denmark). The immune complexes were visualized using appropriate secondary horseradish peroxidase-conjugated antibodies (DAKO, Denmark) at a dilution of 1:10,000 and enhanced chemiluminescence (ECL) Western blotting substrate (Thermo Fisher Scientific, Grand Island, NY, USA). The density of the specific bands was quantified using Image Lab v5.2.1 software (Bio-Rad, Hercules, CA, USA).

The dss1Δ strain has been described before [23 (link)]. The pDUAL vector [26 (link)] was used for the expression of dss1⁺ and the dss1 variants carrying N-terminal HFG (6His, Flag, green fluorescent protein (GFP)) tags. Cloning and mutagenesis were performed using Genscript. The yeast strains were transformed using lithium acetate [27 (link)]. Growth assays were performed on Edinburgh minimal media (EMM2) (San Diego, CA, USA) as described previously [23 (link)]. The preparation of cell lysate samples for SDS-PAGE was performed using trichloroacetic acid and glass beads as described previously [23 (link)]. The samples were separated by SDS-PAGE on 12.5% acrylamide gels and transferred to 0.2 μm pore-size nitrocellulose membranes (Advantec, Tokyo, Japan). The antibody was anti-GFP (1:1000, Chromotek, Planegg, Germany, Cat# 3H9). Secondary horseradish-peroxidase-conjugated antibodies were from Dako Cytomation. Equal loading was checked using stain-free imaging with 0.5% trichloroethanol (Sigma, St. Louis, MO, USA).