br Biospace Lab Paris France br Synthesis of Benp biotin
(Biospace Lab, Paris, France).
2.4. Synthesis of Benp-biotin and Benp-sepharose beads
2-(2-(2-(2-(Piperidin-1-yl) propoxy) benzyl) phenoxy) ethanamine was prepared for synthesis of Benp-biotin and Benp-sepharose beads. The coupling reaction was performed with N-(+)-biotinyl-6-hexanoic Stearamide (Sigma-Aldrich) in DMF solution (Sigma-Aldrich). HATU (Sigma-Aldrich) was added to the solution and stirred for 24 h at room tem-perature. Benp-biotin was purified by HPLC and analysed by mass spectrometry. To prepare Benp-sepharose beads, the ethanamine (60 mg) was dissolved in coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3), and NHS-activated Sepharose™ 4 Fast Flow (GE Healthcare Biosciences, Pittsburgh, PA, USA) were added to the solution. The mixture was incubated in a 4 °C shaker for 24 h. Then, the remaining NHS-activated groups were blocked with blocking buffer (0.5 M etha-nolamine and 0.5 M NaCl, pH 8.3). Benp-conjugated sepharose beads were washed with washing buffer (0.1 M Tris-HCl, pH 8.9).
2.5. Affinity chromatography and pull-down assay
DLD-1 cells were washed with PBS and then homogenized with a sonicator in binding buffer (10 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl2, 1 mM EDTA and 0.1 mM Na3VO4). The cell lysate was cen-trifuged at 13,000 rpm for 30 min at 4 °C, and the supernatant was collected. The cell lysate was pre-cleared by incubation with Sepharose beads (GE Healthcare Biosciences) and then loaded on 15 ml of Benp-tagged Sepharose beads. After washing with 3 bed volumes of washing buffer (0.1 M Tris-HCl, pH 8.9), affinity chromatography was per-formed with pH gradient elution buffer (0.1 M Tris-HCl pH 4.5–6.0). Samples were concentrated, separated by SDS-PAGE, and visualized by Coomassie Brilliant Blue staining. Samples were then digested with trypsin, and a peptide tandem mass spectrometry analysis of the di-gested peptides was performed using an electrospray ionization quad-ruple time-of-flight mass spectrometer.
For pull-down assay, the cell lysate was pre-cleared by incubation with Neutravidin beads (Thermo Fisher Scientific Inc., Rockford, IL, USA) for 30 min at 4 °C. The cleared lysate was incubated with biotin-conjugated Benp (Benp-biotin) overnight at 4 °C. Proteins associated with Benp-biotin were precipitated with Neutravidin beads. After 3 washes in washing buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Tween-20, 10% (v/v) glycerol, 1 mM NaF, 0.1 mM Na3VO4, and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA)), the agarose beads were eluted with elution buffer (0.1 M glycine-HCl, pH 2.8). The samples were boiled in SDS-PAGE sample buffer and separated by 12% polyacrylamide gel elec-trophoresis.
2.6. Molecular docking
To build a structural model of Arpc2 in complex with Benp, we performed molecular docking studies with the LibDock program in Discovery Studio 2.5 software (Accelrys). The Benp ligand was gener-ated using ChemBioDraw and the energy-minimized structure was transferred to Discovery Studio 2.5. The crystal structure of Arpc2 bound to Arp3 obtained from the RCSB Protein Data Bank (PDB code: 1TYQ) was used for docking studies. The number of generated poses was set to 100 for the ligand with default settings for the other para-meters. The structure with the highest LibDock score was selected to be a final structural model. Figures of the complex were drawn using the PyMOL software package.
2.7. Pyrene actin polymerization assay
Actin polymerization assays were performed using the Actin Polymerization Biochem Kit (Cytoskeleton, Inc., Denvor, CO, USA). Biochemical Pharmacology 163 (2019) 46–59
Fluorescence was recorded at an excitation wavelength of 360 nm and an emission wavelength of 410 nm at a constant 25 °C using a Wallac Victor Model 1420–002 fluorescence spectrophotometer (Perkin Elmer, Waltham, MA, USA). The fluorescence data were collected for 1,000 s, and all experiments were performed at least three times with similar results. Pyrene-labelled actin, GST-WASP VCA, and the Arp2/3 complex were purchased from Cytoskeleton, Inc.
2.8. Western blotting
Cell lysates were prepared in RIPA lysis buffer (50 mM Tris, pH 7.0,
150 mM NaCl, 5 mM EDTA, 1% deoxycholic acid, 0.1% SDS, 30 mM Na2HPO4, 50 mM NaF and 1 mM Na3VO4) containing a protease in-hibitor cocktail (Roche Diagnostics). Proteins (20–50 μg) were resolved by 8–15% SDS-PAGE and transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat dry milk or 5% BSA in TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl and 0.1% Tween 20) and incubated with primary and secondary antibodies according to the manufacturer’s protocols. The membranes were detected with the Luminata Forte Western HRP substrate (EMD Millipore) using the LAS 4000 mini (GE Healthcare Biosciences, Pittsburgh, PA, USA). The densitometric analysis of the bands was performed using the MultiGauge program (Fuji Photo Film Co., Ltd., Tokyo, Japan).