Pre stained protein ladder

In order to separate and visualize the laccase isozymes in the culture native, 10% PAGE was used. The electrophoresis was conducted at 4 °C and 145 V. A 10-µg aliquot of the individual protein sample was loaded per gel lane. The laccase activity was visualized with 0.01% guaiacol (Sigma Chemical Co., St. Louis, MO, USA) in 0.1 M citrate-phosphate buffer (pH 5.3), at 20 °C. Simultaneously, 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli [29 (link)]. After separation, proteins were visualized by silver [30 ] or Coomassie Brilliant Blue G-250 staining. Prestained protein ladder (Fermentas, Glen Burnie, MA, USA) was used as a molecular weight marker. A G:Box (Syngene, Frederick, MD, USA) gel documentation system was used for gel imaging.

Cells were treated with various concentrations of SK228 for 24 or 48 h. After treatment, cells were collected and lysed in PRO-PREPTM Protein Extraction solution (iNtRON Biotechnology). The lysates were incubated on ice for 20 min and centrifugated at 13,000 g for 30 min. Protein concentrations were measured by using protein assay reagents (Pierce). Equal amounts of total protein were mixed with 4X sample loading buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 20% SDS, 2.5% bromophenol blue, and 5% 2-mercaptoethanol), boiled for 10 min and then fractionated by using electrophoresis on 8% or 10% SDS-PAGE. A pre-stained protein ladder (Fermentas) was used as the molecular weight standard. Proteins were electrically transferred to PVDF membranes and treated for 1 h at room temperature with 0.05% TBST and 5% non-fat milk. Blots were subsequently incubated at 4°C overnight with primary antibodies. Immunoreactive proteins were detected after incubation with horseradish peroxidase-conjugated secondary antibody (PerkinElmer) for 1 h at room temperature. The immunoblots were visualized by using enhanced chemiluminescence (Perkin Elmer). All antibodies used are provided in Table S1.

The proteins were separated by electrophoresis in the presence of SDS using 10 % polyacrylamide gel under denaturing conditions, according to Laemmli method (Laemmli 1970 (link)). The PageRuler Prestained Protein Ladder (Fermentas, Lithuania) was used as a molecular weight protein marker.

Protein samples were prepared from equal amounts of bacteria cells after overnight growth at 30°C. Bacteria were harvested by centrifugation at 10,000×g for 10 min at 4°C. The culture supernatant fluid was precipitated with 10% trichloroacetic acid (TCA). Briefly, 1 volume (250 µl) of 50% TCA stock was added to 4 volumes (1 ml) of protein sample. The protein-TCA mixture was kept on ice for 15 min, and subsequently the tube was centrifuged at 15,000×g for 5 min. The supernatant was removed and the protein pellet was washed with 200 µl of cold acetone. Finally, the tube was centrifuged at 15,000×g for 5 min and the resulting pellet was dissolved in sample buffer containing 10% glycerol, 0.05% bromophenol blue, 2% SDS, 5% 2-mercaptoethanol, and 10 mM Tris-HCl, pH 6.8. Proteins with known molecular masses (Fermentas) were used as molecular mass markers. SDS-PAGE and Western blotting were carried out according to the methods of Laemmli [56] (link) and Towbin et al. [57] (link). HRP-conjugated donkey anti-rabbit IgG (Promega, USA) was used as secondary antibody. Detection was performed using ECL Prime Western Blotting Detection Reagent (Amersham or GE Life Sciences, USA). Pre-stained Protein Ladder (SM0679, Fermentas) was used as size standards. Gels were stained with Coomassie brilliant blue.

Western blot experiments were performed as described in Molecular Cloning [Green and Sambrook (2012) ]. Materials were used as follows: PVDF membrane (Roth), Super Signal West Femto Maximum Sensitivity Substrate (Termo Scientific), Mini Trans-Blot Cells (BioRad) for wet western blotting, Mini Protean Cells (BioRad) for SDS gel electrophoresis, and Prestained Protein Ladder (Fermentas). Protein lysate was extracted from HEK cell and detected with Anti-HA-Peroxidase (5 mU/ml 1:2500 roth). Each lane is 40x dilution of original lysate by lysis buffer. Relative density of each band is analyzed by ImageJ (1.48v, National Institutes of Health, USA).

Blue native PAGE (3.5–13%) electrophoresis was realized following a procedure described in [66 (link)] using HMW Native Marker Kit (GE Healthcare) for protein standards. SDS-PAGE (8%) was realized according to [67 (link)] using prestained protein ladder (Fermentas) as molecular weight size marker. Both blue native PAGE and SDS-PAGE were revealed using Coomassie blue staining.

WT CXCL12 was chemically synthetized by F. Baleux, Pasteur Institute, Paris and the WT CCL5 was purchased from Immunotools. The molecular weight markers (PageRuler Unstained and PreStained Protein Ladder) were purchased from Fermentas.
E. coli BL21 (DE3) cells transformed with the different expression plasmids were grown at 37°C in Luria Bertani medium supplemented with 100 µg.mL⁻¹ ampicillin until cultures reached an optical density of 0.6 to 0.8 at 600 nm. Then, 1 mM isopropylthio-β-D-galactopyranoside was added to the cultures in order to induce expression of recombinant proteins. Cells were further incubated for 3 h at 37°C (CXCL12-HIS, CXCL12-Strep and CCL5-Strep) or 16 h at 20°C (MBP-CXCL12-Strep, MBP-CCL5-Strep, CXCL12-LT-HIS, CXCL12-LT-Strep and CCL5-LT-Strep) and harvested by centrifugation for 30 min at 5,000 g.