Nonidet np 40

The method of Schreiber and colleagues [14 (link)] was used for the subcellular fractionation of PBMCs, with minor modifications. Ice-cold hypotonic lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1% protease and phosphatase inhibitor cocktail) was used to resuspend cells. Then, they were incubated on ice for 25 min for swelling, after which 25 µL of 10% Nonidet NP-40 (Sigma-Aldrich) was added. Samples were vortexed and centrifuged at full speed to separate the supernatant (containing cytoplasmic proteins) and pellets (containing nuclear proteins). Ice-cold hypertonic nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1% protease and phosphatase inhibitor cocktail) was used to resuspend nuclear pellets, which were incubated on ice for 20 min with agitation. The nuclear extracts were then centrifuged at the maximum speed for 5 min at 4 °C to collect supernatant containing the nuclear proteins. Both cytoplasmic and nuclear extracts were stored at −80 °C until use.

The different Ostreococcus strains were grown in AQUIL medium containing various concentrations of Fe(III)-EDTA, in 24-wells microplates, under 20 µmol quanta m⁻² s⁻¹ of blue light irradiance at 20 °C. After 6 days, cells were harvested in 2 mL micro-tubes by centrifugation at 8 000 × g for 10 min. Dry pellets were frozen in liquid nitrogen ant stored at at −80 °C until extraction. All manipulations were carried out on ice. Cell pellets were ground in 50 µL extraction buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 0.5% (v/v) Nonidet NP40 (Sigma), 10% (v/v) glycerol, using a Tissue Lyser system (Quiagen). The samples were then centrifuged at 10 000 × g for 10 min to remove filter debris. The supernatant was collected, and the total protein concentration was determined using BCA (Bicinchoninic Acid) assay as described elsewhere^{31 (link)}.

Usage of Zebrafish (ZF) embryo experiment was conducted under approved protocol (IACUC/026) by WUHS, Pomona, California. WIK strain of 1 day old Zebrafish embryos were obtained from ZFIN. On day 3 the zebrafish embryos were infected with a mixture containing 10⁸ HSV in 10 μg/mL lipofectamine (10 μg/mL) in 96 well-dishes. In parallel experiments, zebrafish embryos were pre-treated with heparinase I and II (1U/ml for 5 h) or mock treated before HSV-1 infection. Addition of the soluble substrate [o-nitrophenyl-β-D-galactopyranoside (ONPG, ImmunoPure, Pierce; 3 mg/ml)] in the culture medium followed by fluorescence measurement led to the generation of dose–response curves for HSV-1 infected zebrafish embryos. The enzymatic activity was measured at 410 nm using a micro-plate reader. For X-gal assay, after 24 h post-infection, the embryos were fixed (2% formaldehyde and 0.2% glutaraldehyde) overnight at 4°C, permeabilized (2 mM MgCl2, 0.01% deoxycholate, and 0.02% nonidet NP-40 (Sigma) for 8 h at 4°C and then 1 mg/mL X-gal in ferricyanide buffer was added to the embryos and left overnight at 37°C. The embryos were then imaged the next day.

Medium 199 (M199; cat. no. M2154), fetal bovine serum (FBS; cat. no. F9665), insulin (cat. no. I5523), LH (cat. no. L6420), P₄ (cat. no. P8783), PGE₂ (cat. no. P0409), PGF_2α (cat. no. P5069), Laemmli buffer (cat. no. 38733), Tris, KT5720 (product no. K3761), sodium deoxycholate, Nonidet NP-40, sodium dodecyl sulfate (SDS), protease inhibitors, dithiothreitol (DTT), Tween 20, and bromophenol blue were obtained from Sigma-Aldrich (St. Louis, MO, USA). Porcine PNX-14 amide (cat. no. 079-01) for cell culture was bought from Phoenix Pharmaceuticals Inc. (Burlingame, CA, USA). Bovine serum albumin (BSA, cat. no. ALB001) was obtained from BioShop (Ontario, Canada). The Tissue Protein Extraction Reagent (TPER; cat. no. 78510), electrophoresis markers, TaqMan Gene Expression Cells-to-CT Kit (product no. AM1728), Lipofectamine 3000 (product no. L3000001), antibiotic–antimycotic solution, and trypsin (cat. no. 26616) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). PD98059 (cat. no. 1213) was obtained from Tocris (Bristol, UK).

Cells were harvested and plated as described for western blotting. At the time points indicated, cells were trypsinised and transferred to 5 mL BD Falcon tubes (BD Biosciences). Citrate buffer (trisodium citrate (301287F, BDH Laboratory Supplies, Poole, UK), 121 mg Tris Base (T1378, Sigma), 1044 mg spermine tetrahydrochloride (S2876, Sigma) and 2mL Nonidet NP40 (N3516, Sigma) in 2000mL distilled water, pH7.6) was added after centrifugation. The following solutions were added in sequence prior to analysis: 450uL solution A (0.003% trypsin type IX-S (T0303, Sigma) in citrate buffer, pH7.6) for 2 min, solution B (0.05% trypsin inhibitor (T9253, Sigma) and 0.01% RNAse A (R4875, Sigma) in citrate buffer pH7.6) for 10 min, and solution C (0.0416% propidium iodide (81845, Sigma) and 0.1% spermine tetrahydrochloride (S2876, Sigma) in 500 mL citrate buffer pH7.6) for 10 min in the dark. Apoptosis was detected at 24 h using the TACS Annexin V-FITC Kits (R &D Systems) according to the manufacturer's protocol. Flow cytometry was performed using a BD FACSAriaII SORP (Becton Dickinson, Franklin Lakes, NJ). BD FACSDiva software (Becton Dickinson, Version 6.1.2) was used for instrument control and Flowjo software (Version 7.6.5) for Data analysis.

Gast were frozen in liquid nitrogen and proteins were extracted in RIPA lysis buffer [PBS, 0.1% SDS (Promega), 0.5% Sodium Deoxycholate (Sigma-Aldrich),1% Nonidet NP40 (Sigma-Aldrich), 5 mM EDTA (Sigma-Aldrich), 1 mM Na3VO4 (Sigma-Aldrich), 20 mM NaF (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), cocktails of Protease inhibitors (Sigma-Aldrich)]. Proteins were denatured and loaded in a 10% SDS-PAGE gels (10 μg) and further transferred onto nitrocellulose PVDF membranes. Membranes were incubated with antibodies overnight (Table S1) then washed in TBS-Tween 3‰ and further incubated with anti-Rabbit Horseradish Peroxidase conjugated secondary antibody (#172-1019, Bio-Rad). Signals were revealed with Immuno detection kit ECL Luminata Classico (Millipore) and the imager Molecular Image® ChemiDoc™XRS + (Bio-Rad). Proteins quantification was performed by using ImageLab 3.0 (Bio-Rad).

Chinese hamster ovary (CHO-K1) cells grown in a 6-well plates were transfected with 2.5 µg of human or ZF encoded 3-OST isoforms (3-OST-2,or 4), or pCDNA3.1 using LipofectAMINE (Gibco/BRL). At 16 h posttransfection, the cells were replated into 96-well dishes for infection with recombinant virus. After 6-h post infection, β-galactosidase assay were performed using either a soluble substrate o-nitrophenyl-β-D-galactopyranoside (ONPG; ImmunoPure, Pierce) or X-gal (Sigma). For the soluble substrate, the enzymatic activity was measured at 410 nm using a micro-plate reader. For X-gal assay, the cells were fixed (2% formaldehyde and 0.2% glutaraldehyde) and permeabilized (2 mM MgCl2, 0.01% deoxycholate, and 0.02% nonidet NP-40 (Sigma). Finally, 1 mL of β-galactosidase reagent (1.0 mg/ml X-gal in ferricyanide buffer) was added to each well and incubated at 37°C for 90 min before the cells were examined using bright field microscopy under the 20 × objective (Zeiss, Axiovert 100M). Heparinase-II and -III were obtained from Sigma–Aldrich Chemical.