Proteins, antibodies, and cell culture
The recombinant proteins used in this study were provided by Protgen Co., Ltd. (Beijing, China). ES and ZBP-ES were expressed in E. coli, refolded into native forms and purified. The N-terminal site-directed mono-PEGylation was conducted by addition of 20 mmol/L NaBH3CN and incubating the PEG2000 and proteins (PEG2000:protein = 1.5:1) at room temperature for more than 2 h, followed by purification.
We purchased primary antibodies against extracellular regulated protein kinases (Erk), phosphor-Erk, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PEG (Abcam, Cambridge, UK), and turbo green fluorescent protein (tGFP) (OriGene, Rockville, MD, USA). Anti-ES antibody was from our lab stock.
Human microvascular endothelial cells (HMECs) were from our lab stock and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA).
Circular dichroism (CD)
The far-ultraviolet (UV) CD spectra were obtained by a Chirascan™-plus CD Spectrometer (Applied photophysics, Surrey, UK). ES or its variants were diluted in 5 mmol/L Tris–HCl, pH 7.4, to a final concentration of 10 μmol/L. For each protein, data were recorded three times and corrected by subtracting the baseline spectrum of the buffer.
Tryptophan emission fluorescence
Tryptophan emission fluorescence is a good probe to monitor the subtle tertiary structural changes of ES [16]. Proteins (1 μmol/L) in Tris buffer were measured as previously described [17].
Proteolysis assay
ES or its variants (0.5 mg/mL) were incubated with trypsin (0.2 mg/mL) in phosphate buffer saline (PBS) buffer (pH 7.4) at 37 °C [18]. At each indicated time point, reaction solutions were quickly removed and mixed with the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer to stop the trypsin digestion reactions. The samples were subsequently subjected to SDS–PAGE and stained with the Coomassie dye.
Guanidinium chloride (GdmCl)-induced unfolding
ES or its variants (0.8 μmol/L) were incubated in 5 mmol/L Tris–HCl, pH 7.4, containing GdmCl concentrations ranging from 0 to 6 mol/L. After incubation for 24 h at room temperature, GdmCl-induced denaturation was monitored by the tryptophan emission fluorescence intensity at 318 nm [5, 19]. Data were normalized by subtracting the baseline fluorescence intensity of the buffer. The linear extrapolation method was used to evaluate the value of the free change energy in the absence of the denaturant GdmCl (\(\rm\Delta {\text{G}}^{\text{o}}_{{{\text{N}}{-}{\text{U}}}}\)), the apparent slope of the plots (m), and the concentration of GdmCl at the midpoint of the unfolding transition (Cm) as described by Santoro and Bolen [20].
In vitro protein–protein interactions
The nucleolin-tGFP plasmid was transfected into HMECs. Nucleolin-tGFP proteins were immunoprecipitated following the procedure described in our previous study [21]. Subsequently, ES or its variants were incubated with the nucleolin-tGFP beads for 1 h at 4 °C. After washing with the lysis buffer (150 mmol/L Tris, 50 mmol/L sodium chloride, 1% NP40, protease inhibitor mixture, pH 7.4), the bead-bound proteins were subjected to Western blotting.
Internalization assay
As previously described [22], HMECs were treated with 5 μg/mL ES or its variants for 1 h. Subsequently, cells were washed with acidic buffer (pH 3.5) and ice-cold PBS to remove cell surface-binding proteins. The amount of internalized proteins was examined by Western blotting or immunofluorescence.
Western blotting
Protein samples were separated by SDS–PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking for 1 h at room temperature, the membrane was incubated with the indicated primary antibodies overnight and then to the corresponding horseradish peroxidase (HRP)-conjugated goat-anti-mouse or goat-anti-rabbit secondary antibodies for 1 h. The enhanced chemiluminescent substrate was added to the blot, and reactive bands were detected.
Immunofluorescence
Cells were seeded directly on coverslips, fixed, and stained. After blocking with 5% goat serum, cells were incubated with the indicated primary antibodies and fluorescein isothiocyanate (FITC)-labeled secondary antibodies. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a Nikon A1 confocal microscope (Nikon Corporation, Tokyo, Japan).
Transwell migration and wound healing
Transwell migration and wound healing assays were conducted as previously described [22]. Cells were treated with the indicated proteins for 6 and 48 h at 37 °C, respectively. All experiments were repeated twice.
Statistical analyses
All data from the experiments were presented as means ± standard deviations (SDs). Differences between two groups were calculated by GraphPad (GraphPad Software, San Diego, CA, USA), and considered significant if the P value was <0.05 using a two-tailed Student’s t test.