Fig 1: The ANC count versus time profiles obtained by subcutaneously administered recombinant proteins.Each rat in the respective group received injections of SCF-La-GCSF (500 or 1,000 µg/kg), G-CSF (500 µg/kg), SCF (500 µg/kg), a mixture of SCF and G-CSF (500 + 500 µg/kg), and a control buffer. Results are expressed as the mean ± SEM. Individual values from the independent assays are indicated by the open circles. A one-way ANOVA combined with the Tukey’s multiple comparison test was performed to compare the means of ANC values at different time points in each group (buffer or protein). *Values are significantly different between ANC count induced by injection of each protein at different time points (*p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001).
Fig 2: Purification characteristics of the fusion proteins.(A) A flowchart of the purification steps of SCF-La-GCSF and GCSF-La-SCF. (B) RP-HPLC of the purified fusion proteins. The SCF and G-CSF monomers were used as controls. Fifteen-µg of each protein was loaded onto a Zorbax 300SB-C18 column. Absorbance at 215 nm is reported as AU. (C) SDS-PAGE of the purified fusion proteins under non-reducing and reducing conditions. Lanes 1, 4, SCF-La-GCSF; lanes 2, 5, GCSF-La-SCF; lane M, prestained molecular weight marker (Thermo Fisher Scientific, Waltham, MA, USA).
Fig 3: SE-HPLC analysis of purified SCF-La-GCSF and GCSF-La-SCF.Ten-µg of each protein was loaded onto a TSK-gel G3000 SWXL column. The column was calibrated with a protein standard mix (Sigma–Aldrich, St. Louis, MO, USA). The SCF and G-CSF monomers were used as controls. Absorbance at 280 nm is reported as AU.
Fig 4: The proliferation of G-NFS-60 (A) and M-07e (B) cells induced by the purified SCF-La-GCSF and GCSF-La-SCF.The plots represent the specific biological activity of the heterodimers calculated from proliferation curves using the equations as described previously (Mickiene et al., 2017). Results are expressed as the mean ± standard deviation (SD). Individual values from the independent assays are indicated by the squares. * Values are significantly different between the biological activity of heterodimer and monomer (the one sample t-test, **p = 0.01 and ****p = 0.0001). Biological activity of G-CSF and SCF are provided by the manufacturers (indicated by the circle and triangle). #Values are significantly different between the biological activity of SCF-La-GCSF and GCSF-La-SCF (the unpaired t-test, ##p = 0.01 and ####p = 0.0001).
Fig 5: Identification of the purified fusion proteins with the antibodies in Western blot.The proteins were separated on a 15% SDS-PAGE under non-reducing and reducing conditions. (A) The Western blot with the monoclonal antibody against G-CSF. Lanes 1, 4, the G-CSF monomer; lanes 2, 5, SCF-La-GCSF; lanes 3, 6, GCSF-SCF. (B) The Western blot with polyclonal antibodies against SCF. Lanes 1, 4, the SCF monomer; lanes 2, 5, SCF-La-GCSF; lanes 3, 6, GCSF-La-SCF. Lane M, prestained molecular weight marker (Thermo Fisher Scientific, Waltham, MA, USA).
Supplier Page from Abcam for Recombinant human SCF protein (Animal Free)