N dodecyl β d maltoside ddm

HEK293T cells were transfected using PEI 2500 (Polysciences). Cells were passed the day before transfection in DMEM media (Gibco), 10% fetal bovine serum (Invitrogen), 1% Penicillin-Streptomycin (Gibco) in 10 cm dishes. On the day of transfection cells were 60–70% confluent. The medium was refreshed 2 h before transfection; 5 µg DNA was mixed with 250 µL PBS, after which 35 µL PEI 2500 was added to the DNA-PBS mixture. The transfection mixture was gently vortexed, incubated for 10 min at RT and drop-wise added to HEK293T cells. After transfection (48 h), cells were harvested in 1 mL of lysis buffer (25 mM HEPES/NaOH, pH 7.4, 150 mM NaCl, EDTA-free protease inhibitor cocktail (Roche)) with 1% n-dodecyl β-d-maltoside (DDM) (Thermo Scientific) or 1% Triton X-100 (Roche, for PICK1), incubated (45 min, rotating) at 4°C, spun down at 20,800 g for 10 min at 4°C. The obtained supernatant was used for co-immunoprecipitation.

Chemicals for cultivation of Escherichia coli and P. chrysosporium DSM 1556, including yeast extract, malt extract, soytone, lysogeny broth, TRIS, MES, and salts for minimal media were obtained from Carl Roth (Germany). Beech wood chips for growth were obtained from J. Rettenmaier Söhne GmbH & Co. KG. (Germany). Carboxymethyl cellulose (CMC), lichenan, mannan, xyloglucan, and glucomannan were purchased from Sigma Aldrich (USA), and beech wood xylan was purchased from Carl Roth (Germany). para-nitrophenol (pNP), para-nitrophenyl-β-d-galactopyranoside (pNP-Gal), para-nitrophenyl-acetate (pNP-acetate), para-nitrophenyl-β-d-glucopyranoside (pNP-Glc), para-nitrophenyl-β-d-xylopyranoside (pNP-Xyl), para-nitrophenyl-β-d-mannose (pNP-Man), para-nitrophenyl-β-d-arabinofuranoside (pNP-Ara), and para-nitrophenyl-N-acetyl-β-d-glucosamine (pNP-GlcNAc) were purchased from Megazyme (Ireland), n-dodecyl β-d-maltoside (DDM) was obtained from Thermo Scientific (USA) and bovine serum albumin (BSA) from VWR Chemicals (USA). The sources of supply of more methodology-specific reagents are reported in the corresponding procedure section.

Transfected cells were lysed on ice for 15 min in a buffer (100 mM NaCl, 1 mM TCEP [Tris (2-carboxyethyl) phosphine hydrochloride], 2× protease inhibitor, and 10 mM HEPES; pH 7.5) containing 1% n-dodecyl-β-D-maltoside (DDM; Thermo Scientific). The resultant samples were resuspended in 1× Laemmli buffer containing 5% β-mercaptoethanol (Bio-Rad), boiled for 10 min, and subjected to protein separation by SDS-PAGE in 4–20% Mini-PROTEAN TGX precast gels (Bio-Rad) before transferred to nitrocellulose membranes (Wako). The membranes were incubated in a blocking buffer (Nacalai Tesque) for 1 h at room temperature and then mixed with primary antibodies, including rabbit anti-SARS-CoV-2 Spike (S1/S2) polyclonal antibody (1:2000; Invitrogen, Cat# PA5-112048) and mouse anti-β-actin monoclonal antibody (1:5,000; Wako, Cat# 010-27841), followed by staining with the horseradish peroxidase (HRP)-conjugated anti-rabbit (1:50,000; GE healthcare, Cat# NA934VS) and anti-mouse (1:25,000; GE healthcare, Cat# NA931VS) IgG secondary antibodies. The membrane was developed with the ImmunoStar LD enhanced chemiluminescence reagents (Wako) and visualized using ImageQuant LAS 4000 (GE Healthcare).