Anti rabbit igg

HEK 293T were lysed with NP-40 buffer (20 mM Tris-HCl pH 7.5, 10% glycerol, 137 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, 10 mM sodium fluoride, EDTA 2 mM) containing protease inhibitors (Sigma). Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Biorad). The membranes were incubated with mouse monoclonal anti-EBV EBNA-1 antibody (Santa Cruz) and rabbit polyclonal anti-αTubulin (ABclonal), followed by HRP-conjugated anti-mouse (Santa Cruz) or anti-rabbit IgG (DAKO).

The amount of Pr55 or p24 protein in lysates was quantified with the aid of an Enzyme-linked Immunosorbent Assay (ELISA) using MAb M01 (1:1000, Polymun, Vienna, Austria) as coating antibody [22 (link)]. An ELISA with gp41-derived peptides was used to quantify anti-gp41 immunoglobulins in animal sera by the end-point dilution method in duplicates [23 (link)]. Peptides spanning the MPER (EQNEKDLLALDSWNNLWNWFDITKWLWYIK) and CHR regions (MQWDREISNYTNTIYRLLEDSQSQQEQNEK, both from Pepscan Presto BV, Lelystad, Netherlands) were used for coating at 100 ng/ml on Nunc Maxisorp plates (Thermo Fisher Scientific, Waltham, USA). HRP-coupled polyclonal anti-rabbit-IgG and anti-human-IgG served as secondary antibodies (1:4,000, Dako). Washing was done with the aid of a high-throughput microplate washing device (MAP-C2 workstation, Titertek Instruments Inc., Huntsville, USA).

Anti-IRE1α was prepared as follows. cDNA encoding the cytosolic domain (1513–3054) corresponding to amino acids 465–977 of mouse IRE1α (GenBank Accession No. NM023913) was amplified using pcDNA3.1 (+) mIRE1α 33 (link) as a template and 5′-ACATGCATGCACTTACCCCCTGAGCGTG-3′ and 5′-ACGCGTCGACTCAGAGGGCATATGGAATCACT-3′ as primers. The amplified DNA was digested with SphI and SalI and cloned into a pQE80L vector (Qiagen, Hilden, Germany) to produce the His-tagged protein in Escherichia coli. Antigens were purified as previously described34 (link). For immunization, a rabbit was injected with an emulsion containing 0.2 mg antigen and Sigma Adjuvant System (Sigma-Aldrich, St. Louis, MO), for 4 times with 3-week intervals between injections; antiserum was collected at 10 days after the final injection. Anti-C/EBP homologous protein (CHOP), anti-eukaryotic initiation factor 2α (eIF2α), anti-phospho-eIF2α, and anti-β-actin were purchased from Affinity BioReagents/Thermo Fisher Scientific (Waltham, MA), Cell Signaling Technology (Danvers, MA), Biosource (Camarillo, CA), and Novus Biologicals (Littleton, CO), respectively. Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from DAKO (Glostrup, Denmark) and GE Healthcare (Little Chalfont, UK), respectively.

Protein lysates were prepared from basal ganglia and SNpc of human brains from PD, DS, and age-matched controls (n = 6 in each group) as described previously [26 (link)]. 20 μg protein samples were separated on 15–20% Bis-tris gels and transferred to 0.45 or 0.2 μm pore size PVDF membranes (Invitrogen). The membranes were incubated with the appropriate primary antibody in blocking buffer for 24 hour at 4 °C and then washed three times with 0.1M tris saline buffer with 1% Tween 20 (TBST) followed by incubation for 1 h at room temperature (RT) with HRP-conjugated secondary antibodies (anti mouse IgG 1:3000, DAKO, Carpinteria, CA, USA) or anti-rabbit IgG (1:3000; DAKO, USA) antibodies. Binding was detected with ECL Plus chemiluminescence reagents and Hyperfilm ECL (both from GE Healthcare, Chicago, IL, USA).

Protein samples were collected in 2 × Laemmli buffer (Bio-Rad) containing 5% β-mercaptoethanol (Sigma) and run in 4–20% Mini PROTEAN TGX Precast Protein Gel with 10 50 µl wells (Bio-Rad). The proteins were blotted from the gel to PVDF membrane using Trans-Blot Turbo Transfer System (Bio-Rad) and Trans-Blot Turbo RTA Mini PVDF Transfer Kit (Bio-Rad). Mouse anti-β-actin (1:1000; Santa Cruz; sc-47778), rabbit anti-HIF1α (1:1000), rabbit anti-cleaved caspase-3 (1:500; Abcam; ab32042), mouse anti-MyBPC3 (1:500) and mouse anti-Troponin T (1:1000; Abcam; ab33589) were used as primary antibodies and horseradish peroxidase-conjugated anti-mouse IgG (1:3000; Santa Cruz; sc-516102) and anti-rabbit IgG (1:2000; Dako; P0217) as secondary antibodies (full protocol in Supplementary Information). The protein-antibody complexes were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) and ChemiDoc MP Imaging System (Bio-Rad) was used for imaging. The images were analyzed using Image Lab Software (Bio-Rad).

Proteins were extracted from previously frozen lungs. A Compartmental Protein Extraction Kit (Chemicon International, Temecula CA) was used. 200–400 mg tissue was homogenised in cold buffer C (1 ml/g tissue) and Ultra Turrax (KA, Staufen, Germany). Two protein fractions were obtained for each sample: cytoplasm and membrane, and they were quantified. Proteins in each fraction (100 mg) were separately run on a Tris-HCl/SDS gel, 8% acrylamide, and they were transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham). After washing the membrane with distilled water, it was blocked with a PBS/Tween solution, 0.2%, with 5% skimmed milk. It was then incubated with the primary antibody during 2 hours at room temperature. The antibodies used were Anti-Rat AQP1 (Alpha Diagnostic, San Antonio, TX) and Anti-Rat AQP5 (Alpha Diagnostic, San Antonio, TX), both with a 2 mg/ml concentration. After several washes with PBS/Tween 0.2%, it was incubated with the secondary antibody Anti-Rabbit IgG (DAKO, Glostrup, Denmark) in 1∶2000 dilution. β-actin expression was detected as an internal control, and relative protein content was analysed using the enhanced chemoluminiscence method.

Fetal-membranes were collected after delivery and processed for histological analysis. Tissues were fixed in 10% formalin/PBS and embedded in paraffin, then 10 μm-thick sections were cut and fixed on New Silane III glass slides (Muto Pure Chemical, Tokyo, Japan). Immunohistochemical staining was performed with a Benchmark GX system (Ventana, Tucson, AZ, USA). The following primary antibodies were used: mouse anti-COX-1 (sc-19998, 1:30 dilution; Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-COX-2 (sc-166475, 1:150; Santa Cruz Biotechnology); mouse anti-p23 (ab92503, 1:500; Abcam, Cambridge, MA, USA); and rabbit anti-SLCO2A1 (bs-4710R, 1:250; Bioss Antibodies, Woburn, MA, USA). Secondary antibodies were anti-rabbit IgG (1:3,000; DAKO, Santa Clara, CA, USA) and anti-mouse IgG (1:3,000; DAKO). The OptiView Amplification Kit and OptiView DAB IHC Detection Kit (Ventana) were used for detection. Mouse monoclonal immunoglobulin (Ventana) was used as a negative control. Hematoxylin was used for nuclear counterstaining, and the images were taken with a light microscope (BZ9000, Keyence, Osaka, Japan).