Hrp conjugated anti rabbit antibody

A. aegerita fruiting bodies (3.5 g), single parts (cap with gills, stem and ring; 3.5 g), or the mycelium (1.5 g) were homogenized by T 10 basic ULTRA-TURRAX (IKA^®- Werke GmbH & Co. Staufen, Germany); for 5 min at 4 °C in 5 mM Na-phosphate, pH 7.2 containing 0.14 M NaCl, while PDB medium was concentrated in an Amicon cell concentrator (MWCO 3 kDa; Sigma-Aldrich) for further analyses. After clarification (60 min at 15,000× g at 4 °C), the protein content of samples was determined by the method of Bradford using Bio-Rad protein assay (Bio-Rad, Milan, Italy). Total protein extracts (10 μg) were separated by SDS-PAGE on a Mini-Protean II mini-gel apparatus (Bio-Rad), using 6% (w/v) stacking polyacrylamide gel and 15% (w/v) separation gel. Separated proteins were transferred onto nitrocellulose membrane (filter type 0.45 μm HATF; Millipore, Burlington, Massachusetts, USA) by electroblotting with Mini Trans-Blot Cell (Bio-Rad). Finally, the blot was probed with the anti-Ageritin polyclonal rabbit antibody (Bio-Fab research, Rome, Italy) as a primary antibody (dilution 1:1000). Following incubation with the anti-rabbit HRP-conjugated antibody (Bio-Rad, 1:3000), protein bands were revealed by adding the Clarity^TM Western ECL substrate (Bio- Rad) and acquired by using the ChemiDoc XRS System (Bio-Rad).

Quinoin or immunoconjugate purity and integrity were determined by SDS-PAGE with a Mini-Protean II mini-gel apparatus (Bio-Rad, Rome, Italy) using 6% stacking and 15% separation polyacrylamide gel; precision plus protein kit (Bio-Rad, Hercules, CA, USA) was used for reference proteins. Protein concentration was determined by Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Rodano, Italy), using BSA as standard. Western blot analyses were performed after SDS-PAGE separation, transferring proteins onto nitrocellulose membrane (filter type 0.45 µm HATF; Millipore, Burlington, MA, USA) by electroblotting with Mini Trans-Blot Cell (Bio-Rad, Hercules, CA, USA). Finally, the blot was probed with anti-quinoin polyclonal rabbit antibody (Bio-Fab research, Rome, Italy) as a primary antibody (dilution 1:2500). Immunoreactive bands were detected following incubation with the anti-rabbit HRP-conjugated antibody (Bio-Rad, 1:3000), by adding the Clarity^TM. Western ECL substrate (Bio-Rad, Hercules, CA, USA) and acquired by using the ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA).

Cells were washed twice with ice-cold PBS, harvested by scraping and whole cell lysates were prepared in RIPA buffer containing phosphatase and protease inhibitors (#SC-24949; Santa Cruz Biotechnologies). Cell lysates were incubated on ice for 30 min and clarified by centrifugation at 20,000 g for 30 min. Protein content was estimated using Bio-Rad protein Assay Dye (Catalog#500–0006). Equal amounts of protein (40 μg) were resolved in 12% SDS-PAGE gels, blotted onto PVDF membranes (#IPVH00010; EMD Millipore) and probed with antibodies for MDR1(D3H1Q; 1:1000; Cell Signaling), MRP1(D708N; 1:1000; Cell Signaling), ABCG2 (D5V2K; 1:1000; Cell Signaling), phospho-p38MAPK (D3F9; 1:1000; Cell Signaling), p38MAPK (D13E1; 1:1000; Cell Signaling), phospho-p44/42 MAPK (Erk1/2) (D13.14.4E; 1:1000; Cell Signaling), p44/42 MAPK (Erk1/2) (137F5; 1:1000; Cell Signaling) phospho-SAPK-JNK (81E11;1:1000; Cell Signaling), SAPK-JNK (#9252; 1:1000; Cell Signaling) and GAPDH (14C10; 1:1000; Cell Signaling). Secondary HRP-conjugated anti-rabbit antibody (#170–6515; 1:1000; Bio-Rad) was used. The antigen-antibody complexes were detected by chemiluminescence using Super Signal West Pico Chemiluminescent Substrate (#34080; Thermo Scientific, USA). Densitometry was performed using Image J software (NIH) and histograms showing relative expression of MDR1, MRP1 and ABCG2 normalized to GAPDH were generated.

Glycosylated or de-glycosylated HA or BSA as a control was coated on a HB 96 well microtiter plate overnight at 4°C using 0.1M carbonate/bicarbonate buffer. Wells were washed with PBS and blocked with 5mM HEPES at pH 7.4 with 150 mM NaCl and 5 mg/mL of BSA. Glycosylated or de-glycosylated SP-A were added at concentrations of 10, 25, 50, 75 and 100 μg/mL in blocking buffer with 2 mM Ca⁺² and incubated for 1 hour at 37°C. After washing, plates were incubated with 1:5000 dilutions of rabbit anti-SP-A polyclonal antibodies followed by washing and incubation with 1:10,000 dilution of an HRP-conjugated anti-rabbit antibody (Bio-Rad Cat No 170-6515). Bound protein was visualized calorimetrically at 450 nm using tetraethyl benzidine (R&D Systems, Cat No DY999) as the HRP substrate and color development stopped using 2M H₂SO₄. Plates coated with albumin were used as controls.

Total T cells were purified by negative selection using BioMag Goat anti-mouse IgG and anti-rat IgG (QIAGEN GmbH, Hilden, German) after 2.4G2 treatment, and incubated with a biotin-conjugated anti-CD3 mAb (2C11, 10 μg/ml, BioLegend, San Diego, USA) for 30 min on ice. Cells were then incubated with streptavidin (20 μg/ml; Sigma-Aldrich Co., St. Louis, USA) for the indicated periods of time at 37°C. For Western blotting, cells were lysed in 5 x lysis buffer containing 125mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.04% bromophenol blue. Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes (ATTO Co., Tokyo, Japan), and probed with anti-ERK1/2 (Cell Signaling Technology, Danvers, USA) or anti-phospho-ERK1/2 (Cell Signaling Technology, Danvers, USA) antibodies followed by an HRP-conjugated anti-rabbit antibody (Bio-Rad Laboratories, Hercules, USA). Signals were detected using an ECL Western blotting kit (GE Healthcare, Buckinghamshire, UK) and Image Quant LAS 4000 (GE Healthcare).