Dithiobis succinimidyl propionate dsp

E. histolytica strain in which the atg8 gene was silenced (atg8-gs) and the control strain (mock vector transfected) were produced from the G3 strain, as described previously (Picazarri et al., 2015 (link)). Trophozoites of these transformants were maintained axenically in Diamond’s BI-S-33 medium (BIS) (Diamond et al., 1978 (link)) supplemented with 10 μg/mL Geneticin at 35.5˚C. Chinese hamster ovary (CHO) cells were maintained in F12 medium (Sigma-Aldrich, St Louis, MO) supplemented with 10% fetal calf serum (Sigma-Aldrich, St Louis, MO) at 37˚C with 5% CO₂. Paramagnetic Dynabeads, dithiobis succinimidyl propionate (DSP), and OPTI‐MEM I medium were purchased from Thermo Fisher Scientific (Waltham, MA). A complete mini mix was purchased from Roche (Basel, Switzerland). Human serum was purchased from Sigma-Aldrich (St Louis, MO). Anti-HA (11MO), anti-Myc (9E10), and anti-FLAG (clone M2) monoclonal antibodies were purchased from Covance (Princeton, NJ) and Sigma-Aldrich. The production of rabbit polyclonal antibodies against EhAtg8, EhCS1, and EhCP-A5 were previously described (Nozaki et al., 1999 (link); Picazarri et al., 2008 (link); Nakada-Tsukui et al., 2012 (link)). All chemicals of analytical grade were purchased form Sigma-Aldrich unless otherwise stated.

For NS2/E2 interaction, 1 million electroporated cells were lysed with buffer (50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS). Lysates were cleared by centrifugation at 16,000xg for 10 min at 4°C and were incubated overnight at 4°C with AR3A antibody against HCV E2 or with rabbit NS2 antibody. Protein A/G-coated agarose beads were added to samples for 2h at room temperature. Immune complexes were then washed and eluted with Laemmli buffer for 5 min at 95°C before western blot analysis.
For NS2/NS5A interaction, 1 million electroporated cells were cross-linked with 1mM dithiobis(succinimidyl propionate) (DSP) (ThermoFisher) 30min at room temperature. Tris (pH 7.5) was added up to 200 mM to quench unreacted DSP. Cells were resuspended in lysis buffer (50mM Tris pH 7.4, 150mM NaCl, 1mM EDTA, 0.5% n-dodecyl-β-maltoside) and treated as for NS2/E2 interaction with incubation with rabbit NS2 antibody.

Hek293T cells were transiently transfected with the myc-tagged PHMA Trib2 or PHMA control vectors as previously described^{15 (link)}. The transfected cells were harvested at 24 h and whole cell lysates were prepared using ice-cold Hepes buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 5% glycerol, with protease and phosphatase inhibitors). Crosslinking was performed using dithiobis-succinimidylpropionate (DSP) (Thermo Fisher Scientific) at a concentration of 1.5 mM, and the reaction quenched using Tris (pH 7.4) (50 mM). One milligram of precleared lysates were incubated with Myc9E10 antibody (Santa Cruz Biotechnology) or normal mouse IgG overnight at 4 °C, followed by incubation with Protein A/G UltraLink™ Resin (Thermo Fisher Scientific) for 1.5 h at 4 °C. The samples were washed in Tris buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 5% glycerol, with protease and phosphatase inhibitors), eluted in Laemmli buffer and analysed by western blotting.

In-cell cross-linking was performed using dithiobis [succinimidyl propionate (DSP)] (Thermo Scientific) as previously described (61 (link)). DSP were freshly prepared as a 25-mM solution in dimethyl sulfoxide (DMSO) and diluted to a working concentration of 0.5 mM in PBS. Cells were washed twice with PBS and then incubated with the cross-linker solution for 30 min at room temperature. Then, cells were incubated at room temperature for 15 min with quenching solution (1M Tris-Cl, pH 7.5). Quenching solution was then removed, and cells were washed twice with PBS and cell lysates were prepared for coimmunoprecipitation assay.

Cortisol antibodies and cortisol molecules were procured from Abcam (MA, USA). The TNF-α antibody and antigen, thiol linker used dithiobis(succinimidyl propionate) (DSP) and dimethyl sufloxide were purchased from Thermo Fisher Scientific Inc. (MA, USA). Milipore de-ionized water (conductivity – 18 MΩ cm) was used to prepare the solutions. Nanoporous polyamide membranes were obtained from GE Healthcare Life Sciences (NJ, USA). Pooled human sweat was procured from Lee Biosolutions (MO, USA). No animal or human subjects were tested in this work.

All-trans-retinoic acid (atRA) and Ara-C were purchased from Sigma Chemical Co. (Sigma, Munich, Germany). Dithio-bis succinimidylpropionate (DSP) was purchased from Thermo Fisher Scientific (Waltham, MA, United States). The following antibodies were used: iASPP (Sigma, 1:2000), Flag (Proteintech Group, Chicago, United States 1:2000), Myc (Proteintech, 1:2000), His (Proteintech, 1:2500), pan-phospho-serine antibody (Santa Cruz Biotechnology, Santa Cruz, CA, 1:2000), GFP (Cell Signaling Technology Inc., Beverly, MA, United States, 1:2000) and Tubulin (Sigma, 1:5000).