Halt protease inhibitor

Cellular thermal shift assays (CESTA) were performed as previously described^{93 (link)} with some modifications. Briefly, approximately 5 × 10⁹ VIT-HA expressing parasites were collected, split into two samples and incubated at 37 °C for 1 h in 800 μl Ringer’s solution ((115 mM NaCl, 3 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 3 mM NaH₂PO₄, 10 mM HEPES, 10 mM glucose)), (untreated) or Ringer’s solution containing 2 mM ferric ammonium chloride (FAC). Cells were then divided into 9 PCR tubes and incubated at the following temperatures for 3 min, (37, 41, 43, 47, 50, 53, 56, 59, 63 °C). Parasites were moved onto ice, 16 μl of 6x lysis buffer (final concentration of 0.8% IGEPAL/NP-40, 1x Halt protease inhibitor (Sigma)) added and cells incubated for 10 min on ice. To ensure full lysis, cells were frozen on dry ice and thawed at RT three times. To remove protein aggregates, cells were spun at 14,000 × g for 80 min at 4 °C, the supernatant was carefully removed and western blotted, as described below.

HeLa cells were plated in 48-well plates to confluence. Particles (7 × 10⁸) of fcHPV16 EdU or uncleaved HPV16 Myc-16L2-HA were added to cells and allowed to bind at 4°C for 1 h. Unbound virus was removed, and cells were incubated at 4°C for 1 h with or without 10 µM HD5, 20 µM NH₄Cl, or 5 µM MG132 alone or in combination. Samples were shifted to 37°C for the indicated times, collected by washing with cold PBS, and then lysed with NP-40 buffer (150 mM NaCl, 1% NP-40, 10 mM Tris, pH 8.0) and HALT protease inhibitor (Sigma). Heat-denatured and reduced samples were run on 10% SDS-PAGE gels and transferred to nitrocellulose. Immunoblots were probed for L1 using anti-CamVir (1:1,000; Millipore), for L2 with anti-HA (1:1,000; Thermo Scientific), or using anti-β-actin (1:5,000; Sigma) as a loading control. Anti-mouse secondary antibodies were conjugated to horseradish peroxidase (HRP) (1:5,000; Thermo Fisher), and signal was developed with enhanced chemiluminescence (Bio-Rad).

Resected cecal tissue was bead beaten for 1 min in 250 μl of lysis buffer I (1× HALT protease inhibitor (Pierce), 5 mM HEPES). Lysis buffer II (250 μl) was added (1× HALT protease inhibitor, 5 mM HEPES, 2% Triton X-100 (Sigma-Aldrich)) and the tubes were inverted gently. Tissue samples were incubated on ice for 30 min, followed by a 5 min spin at 13,000 x g at 4°C. The supernatant was removed to a fresh tube, and total protein concentration was assessed by a BCA assay according to the manufacturer’s instructions (Pierce). IL-1β was measured using Ready-Set-Go! ELISA kit (eBioscience). CXCL1, CXCL2 and IL-25 were measured using R&D Systems Duoset ELISA kits. All procedures are performed according to the manufacturers’ instructions. All data were expressed relative to total protein concentration.

The processing of nuclear and cytoplasmic fractions was performed as previously described (83 (link),84 (link)) Specifically, HeLa cells were washed with 1× PBS (137 mM NaCl (Sigma S9888), 2.7 nM KCl (Sigma P4505), 10 mM Na₂HPO₄ (Sigma S3264) and 2 mM KH2PO4 (Sigma P9791), pH  7.4) and harvested using 500 μl of TLB (50 mM Tris, 150 mM NaCl, 10 mM EDTA, TritonX-100 0.5% v/v, Halt Protease inhibitor 10 μl/ml, phosphatase inhibitors 2 and 3 (Sigma), pH  7.2) buffer per T75 flask. The samples were centrifuged at 13 000 rpm for 15 min at 4°C. The supernatant was transferred to a new microcentrifuge tube (this is the cytoplasmic fraction). The remaining nuclear pellet in the microcentrifuge was gently washed three times with 200 ul of TLB buffer. The nuclear pellets were resuspended in TLB and the nuclear lysate samples were sonicated with a Microson XL-2000 sonicator (Misonix) 4× (6 s sonication/10s rest on ice). Samples were centrifuged at 21 130g for 15 min at 4°C. The resulting supernatant (the nuclear fraction) was then transferred to a new microcentrifuge tube. The protein concentration for each sample was determined using 595 nm wavelength OD values against a bovine serum albumin (BSA) standard.

Cells were washed with 1X PBS (137 mM NaCl (Sigma S9888), 2.7 nM KCl (Sigma P4505), 10 mM Na2HPO4 (Sigma S3264) and 2 mM KH2PO4 (Sigma P9791), pH = 7.4) and harvested using 300 µL of TLB SDS (50 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% sodium dodecyl sulfate, TritonX-100 0.5% v/v, Halt Protease inhibitor 10 µL/mL, phosphatase inhibitors 2 and 3 (Sigma, Livonia, MI, USA), pH = 7.2) per T25 flask. The total lysate samples were sonicated three times for 10 s at 12 watts RMS using a 3 mm wide Qsonica Microson homogenizer with Microson ultrasonic cell disruptor XL2000 (Microson, Brussels, Belgium). The samples were centrifuged at 21,130× g for 15 min at 4 degrees Celsius. The resulting supernatant was transferred to a new microcentrifuge tube. The protein concentration for each sample was determined using 595 nm wavelength OD values against a Bovine Serum Albumin (BSA) standard. An equal amount of protein (15–30 micrograms) was loaded for each sample.

293T cells were lysed 48 h posttransfection in 160 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.25% NP-40, supplemented with Halt Protease Inhibitor, 1 mM PMSF (Sigma), and RNaseOUT (Life Technologies). RNase inhibitors were omitted from samples treated with 15 μg/ml RNaseA (Life Technologies). FLAG- or T7-tagged L1 proteins were immunoprecipitated with anti-FLAG M2 (Sigma) or anti-T7 (Novagen) monoclonal antibodies coupled to magnetic beads (Dynabeads, Thermo Fisher). Precipitated proteins were separated by SDS page and transferred onto PVDF membranes and probed with anti-myc (9B11) antibody (Cell Signaling) anti-FLAG M2 (Sigma), or anti-T7 antibody (Novagen). Next, membranes were incubated with anti-mouse HRP light chain-specific or anti-rabbit HRP conformation-specific secondary antibodies (Cell Signaling). For immunoprecipitation of SAMHD1 combined with subsequent LEAP reaction (LEAP-IP), 293T cells transfected with pAD2TE1 and SAMHD1-myc or empty vector were lysed and myc-tagged SAMHD1 was precipitated using anti-myc antibody bound to magnetic beads (Cell Signaling). The beads harboring SAMHD1 and L1 RNPs were eluted in MLV-RT buffer (Promega). Samples for LEAP reaction were directly subjected to reaction, samples for MLV-RT reactions were incubated for 10 min at 95 °C. LEAP and MLV-RT reactions were performed as described above.

Recombinant SmSoxS1 was produced, expressed, and purified as a Fusion protein with Maltose binding protein (MBP) using the pMAL Protein Fusion and Purification System (NEB, Ipswitch, MA, USA). SmSOXS1 was subcloned into pMAL-c5X (NEB, Ipswitch, MA, USA), and this plasmid construct was transformed and induced in BL21 (DE3) E. coli cells (Invitrogen, Waltham, MA, USA) with 0.4 mM IPTG. Cells were disrupted by sonication in lysis buffer (50 mM potassium phosphate pH 8.0, 200 mM sodium chloride; Halt protease inhibitor (Thermo Scientific, Waltham, MA, USA); PMSF). Cleared supernatant was incubated with amylose–agarose beads (NEB, Ipswitch, MA, USA) with agitation overnight at 4 °C. The fusion protein was eluted from the beads using a maltose elution solution (50 mM potassium phosphate pH 8.0, 200 mM sodium chloride, 10.4 mM maltose) and quantified using the BCA Protein Quantitation kit (Thermo Scientific, Waltham, MA, USA). Schistosomula samples were disrupted by bead beating with glass beads (Sigma, Burlington, VT, USA) and sonication in a Tris-HCl lysis Buffer (25 nM Tris-HCl pH 7.5, 1 mM DTT, 1X Halt protease inhibitor, 1 mM PMSF). Protein was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA).