Temperature-dependent changes in circular dichroism spectrum from 190 to 250 nm of (A) rhSCF^1–141 and (B) rhSCF^1–165.

<p>Conditions: 300 µg/mL in 100 mM phosphate buffer (pH 7.0); 1, 25°C as control; 2, 70°C for 10 min; 3, 90°C for 15 min; 4, 90°C for 30 min; and 5, 90°C for 60 min.</p

Thermostability study of SCF^1–141 and SCF^1–165 by molecular modeling simulation.

<p>(A) Potential energy of SCF^1–141 and SCF^1–165. The root mean square deviation (RMSD) of SCF^1–141 and SCF^1–165 at (B) 25°C; (C) 70°C; and (D) 90°C. (E) Snapshots of the evolving SCF^1–141 structure at different time points at the indicated temperatures.</p

The effect of temperature and time on the viability of TF-1 cells treated with rhSCF¹⁴¹ or rhSCF¹⁶⁵.

<p>(A) The effect of temperature on the viability of TF-1 cells treated with rhSCF^1–141 or rhSCF^1–165. TF-1 cells were incubated with 100 ng/mL rhSCF^1–141(solid circle) or rhSCF^1–165 (hollow triangle) in PBS (pH 7.4) for 10 minutes and the cell viability assayed. (B) Thermostability of rhSCF^1–141 and rhSCF^1–165 over time as indicated by cell viability. TF-1 cells were incubated with 100 ng/mL rhSCF^1–141(solid circle) or rhSCF^1–165 (hollow triangle) in PBS (pH 7.4) at 90°C for the times indicated. Results are expressed as mean ± SD (n = 3). *P<0.05; **P<0.01; ***P<0.001</p

Effects of rhSCF^1–141 and rhSCF^1–165 on downstream signaling targets MAPK and Akt.

<p>Cells were incubated with rhSCF^1–141 or rhSCF^1–165 in PBS (pH 7.4) at the concentrations indicated. A and B are Western blots using the indicated antibodies. Similar results were obtained in 3 independent experiments. C is the quantitation results of the Western blots shown in A and B.</p

The expression and purification of rhSCF^1–141 and rhSCF^1–165.

<p>(A) PCR analysis of pPICZαC-rhSCF^1–141 (1011 bp) and pPICZαC/SCF^1–165 (1083 bp) using <i>5′AOX I</i> and <i>3′AOX I</i> primers (arrow). (B) Coomassie blue-stained SDS-PAGE of rhSCF^1–141 and rhSCF^1–165overexpressed from <i>Pichia</i> culture supernatants. Lane M: molecular weight markers. Numbers above the lanes represent hours after methanol induction. Arrows indicate the overexpressed rhSCF^1–141 or rhSCF^1–165 (C) Silver-stained 15% SDS–PAGE of purified rhSCF^1–141 and rhSCF^1–165 produced in <i>P. pastoris</i>. Lane M, molecular weight markers. Lanes 1 and 2, purified rhSCF^1–141 (200 and 100 ng, respectively); Lanes 3 and 4, purified rhSCF^1–165 (20 and 50 ng, respectively). (D) Western blot analysis of rhSCF^1–141-6His and rhSCF^1–165-6His from culture supernatants. The recombinant proteins were 6xHis tagged and detected with anti-His antibodies.</p

SDS-PAGE analysis of limited proteolytic digestion of rhSCF^1–141 and rhSCF^1–165.

<p>Purified (A) rhSCF^1–141 or (B) rhSCF^1–165 (15 µg each in 0.1 M-Tris/HCl buffer, pH 8.2) was incubated with trypsin at 25°C or 90°C for 10 minutes (lanes 2 and 3), or preheated at 90°C 10 minutes (lanes 4 and 5), 90 minutes (lanes 6 and 7), or 120 minutes (lanes 8 and 9) and then treated with trypsin for 10 minutes at 25°C or 90°C as indicated above. Lanes 1 and 10 are rhSCF and trypsin only.</p