Alexa fluor 488 nhs ester

The Site 2 and Sites 2,4 peptides were fluorescently labeled at the amidated C-terminus. Both peptides were labeled with the donor probe Alexa Fluor 488 NHS Ester, whereas the Site 2 peptide was additionally labeled with the acceptor probe Alexa Fluor 568 NHS Ester (Thermo Fisher Scientific). The labeling reaction was performed at 25 °C for 1 h in 20 mM MES pH 6.8 and 50 mM NaCl and at a peptide concentration of 5 mg mL⁻¹ with a 2-fold molar excess of fluorescent dye. The labeling reaction was stopped with the addition of a 10-fold molar excess of hydroxylamine (Sigma-Aldrich Corporation). Labeled peptides were separated from any remaining unlabeled peptide using a Symmetry300 C₁₈ reverse phase HPLC column (Waters Corporation, Milford, MA). The peptides were lyophilized, resuspended in 100 mM HEPES pH 7.5, and dialyzed against 25 mM HEPES pH 7.5, 50 mM NaCl and 1 mM MgCl₂. The donor-labeled peptides were pre-incubated with 14-3-3θ at a 1:1 molar ratio (i.e., one peptide per 14-3-3θ dimer). The acceptor-labeled Site 2 peptide (at 5 μM concentration) was then titrated into the preformed complexes. The steady-state fluorescence emission spectra were recorded at 25 °C, with an excitation wavelength of 495 nm (5-nm slit width) and an emission wavelength of 520 nm (5-nm slit width).

The following reagents were purchased: phorbol 12-myristate 13-acetate (PMA) and CP-456773 sodium salt (CRID3) from Sigma-Aldrich (USA); Amicon Ultra 0.5 mL centrifugal filter MWCO 100 kDa, MF-Millipore membrane filters of 0.45 μm and 0.22 μm pore sizes from Millipore Sigma (USA); GM-CSF recombinant human protein, fetal bovine serum (FBS), penicillin-streptomycin, sodium pyruvate (100 mM), GlutaMAX supplement, RPMI 1640 medium, PBS buffer, Pierce LDH cytotoxicity assay kit, cytochalasin D (CytD), Alexa Fluor 488 NHS ester (succinimidyl ester), wheat germ agglutinin Alexa Fluor 555, DRAQ5 fluorescent probe solution (5 mM) and 4% formaldehyde from ThermoFisher Scientific (USA); acridine orange (AO) from Dojindo Laboratories (Kumamoto, Japan); VX765 and lipopolysaccharide (LPS) Escherichia coli O111:B4 from InvivoGen (USA); human IL1 beta assay kit from Cisbio Bioassays (France); CD14 microbeads (human) from Miltenyi Biotech (Germany); silica crystals (MIN-U-SIL-15) from US Silica (USA); μ-Slide 8 well ibiTreat from Martinsried (Germany); THP-1 cells from Japanese Collection of Research Bioresource Cell Bank (Japan); CHO-K1 cells and F-12K medium from ATCC (USA).
Human PBMCs were donated from University Hospital Bonn (Germany), and the operation and usage were approved by the local ethics committee in accordance with the Declaration of Helsinki.