Seeblue plus2

Proteins of crustacean extracts, 15 μg/lane, were separated by electrophoresis under reducing conditions using 4–12% Bis-Tris gels (NuPage, Carlsbad, CA). Pre-stained standards (1x See Blue Plus2, Invitrogen, Carlsbad, CA) were used as molecular weight markers. Proteins were resolved at 200 V for 35 minutes using an Xcell II mini-cell apparatus (Invitrogen) and stained with Coomassie brilliant blue.

To visualise chemokine cleavage, 50 pmol of recombinant human CXCL1 or CXCL8 (R&D Systems) was incubated with C-terminal SpyCEP in a final volume of 20 μl reaction buffer (40 mM Tris-HCL pH 7.5, 0.1% BSA, 75 mM NaCl), at a molar ratio of 1:5, 1:50, 1:500, 1:5000 in favour of chemokine for 2 h at 37 °C. C-terminal^S617A and N-terminal constructs^34–244 were included as controls and assayed at the highest 1:5 molar ratio. Reactions were halted by the addition of Dithiothreitol (DTT) at a final concentration of 100 mM, 4X Bolt LDS sample buffer and heating to 70 °C for 10 min. Samples were separated on pre-cast 4–12% MES buffered Bolt Bis–Tris gradient gels (Invitrogen) by SDS-PAGE gel electrophoresis at 165 V for 35 min with SeeBlue Plus 2 (Invitrogen) used for molecular weight ladder. Gels were stained with PageBlue protein staining solution (Thermo Fisher Scientific) overnight and de-stained in deionised water.

Aliquots (10 μg of total proteins) of rat liver mitochondria prepared as described above were heated to 90 °C for 5 min and electrophoresed into 10% NuPAGE Bis-Tris precast polyacrylamide gels (Invitrogen, Waltham, MA, USA) using a SeeBlue™ Plus2 prestained protein ladder (Invitrogen). The resolved proteins were submitted to immunoblotting and incubated overnight with affinity-purified rabbit antibodies against an N-terminal peptide of rat AQP8 at a final concentration of 1 μg/mL blocking solution (20 mM Tris–HCl, 0.15 M NaCl, 1% Triton X-100, pH 7.5). Horseradish peroxidase antirabbit IgG-treated membranes (antirabbit IgG peroxidase antibody; Sigma) were developed by luminal chemiluminescence (ECL WEST FEMTO PLUS, Immunological Sciences, Rome, Italy). The immunoreactive bands were analyzed by densitometry using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). The density of each band was normalized against that of the housekeeper gene β-actin.

The ten tumor lysates, 20 µg, was diluted in NuPAGE LDS sample buffer (Invitrogen, California, US) with 50 mmol/L dithiotreitol (DTT) and incubated at 95°C for 10 min. Thirty µg of the protein was separated using 4–12% NuPAGE, Bis-Tris, 1 mm thick gels with 15 wells (Invitrogen, California, US) with SeeBlue Plus2 (Invitrogen, California, US) as molecular mass standard. Whole blood from a healthy volunteer (female) was used as a positive control for the rabbit anti-human haemoglobin (A0118, Dako). The electrophoresis was run in MES buffer at 180 V for 1 h and the proteins were then transferred to 0.2 µm PVDF membrane (Trans-Blot Turbo Transfer Pack, Mini format, Bio-Rad) at 25V for 30 min using Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was blocked in 5% non-fat dry milk in 0.2% Tween-20, 150 mM NaCl and 20 mM Tris, pH 7.5 (TTBS) for 3 hours and incubated with rabbit anti-human haemoglobin (10 µg/mL) over night. The membrane was washed three times in TTBS, 10 min each. The membrane was incubated with FITC conjugated polyclonal swine anti-rabbit Immunoglobulins diluted 1∶50, for 1 hour, washed three times, 10 min each, with TTBS and antibody binding was detected with Gel Doc EZ Imager (Bio-Rad). The staining density for each band was analysed with Image Lab Software (Bio-Rad).

Five whole wandering third instar larvae were homogenised in 200 µl lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) and left on ice for 30 minutes. Lysates were standardised by Bradford Assay and heated to 95°C in 2× protein sample buffer (with reducing agent added prior to use) (Invitrogen) and loaded alongside a pre-stained standard (SeeBlue Plus2, Invitrogen) into NuPAGE 4–12% Bis-Tris Protein gel (NP0321PK2). Western blots were transferred onto nitrocellulose membranes and all antibodies were incubated and washed at room temperature in PBS 0.1% Tween-20 and 5% milk. Anti-Tubulin was used as a loading control (T9026 Sigma, 1/1000) to reveal a single 55 kDa band alongside Anti-Syp (McDermott et al., 2012 (link); 1/3000). Quantitative, two-colour Western blot analysis was performed on three biological repeats using a LICOR Odyssey FC instrument and Image Studio V2.0 analysis software.

HEK 293T cells grown in T25 25 cm² flasks were transfected with hTMEM266 and/or hHv1 cDNA using FuGene 6 (Promega). One or two days after transfection, cells were collected in Dulbecco’s PBS and then centrifuged at 500 × g for 3 min. The supernatant was discarded after centrifugation, and a lysis buffer containing 1% Triton X-100 and protease inhibitors (Roche cOmplete tablets) was used to suspend the cell pellet. Cells in lysis solution were sonicated for 5 s on ice and incubated on ice for 30 min, with vortexing for 5 s every 5 min. The cell lysate then was centrifuged at 13,000 × g for 20 min at 4°C, and the supernatant was combined with LDS buffer, DTT, and 2-mercaptoethnol. Samples were heated at 95°C for 5 min and then were centrifuged at 13,000 × g for 2 min. Proteins were separated in a 4–12% NuPage Bis-Tris gel (Invitrogen) using a running buffer containing (in mM): 50 3-(N-morpholino)propanesulfonic acid, 50 Tris base, 3.46 SDS, and 1 EDTA. SeeBlue Plus2 (Invitrogen) was used as the protein molecular-weight marker. Protein in the gel was transferred to nitrocellulose membrane and probed with rabbit polyclonal anti-hTMEM266 antibody custom raised against mTMEM266 residues 524–538.