Ies and oxidative stress.Figure 2. Expression, purification (A) and polycnonal antibody generation (B) of 30Kc6 protein in E. coli. The 30Kc6 gene was inserted into prokaryotic expression plasmid pET-28a and was expressed in E. coli BL21 (DE3) with the induction of IPTG. M. represents the protein molecular weight marker. 1. Empty vector before inducing; 2. Empty vector after inducing; 3. Recombinant bacteria before inducing; 4. Recombinant bacteria after inducing (IPTG 0.1 mmol/L); 5. Recombinant bacteria after inducing (IPTG 1 mmol/L); 6. Purified 30Kc6 protein; 7. Purified antibody reacts with purified 30Kc6 protein; 8. Pre-immune rabbit serum reacts with purified 30Kc6 protein. The arrows indicate the 30Kc6 protein. doi:10.1371/journal.pone.0068746.gPurification and Identification of the Silkworm Protein 30Kc6 Expressed in BmN BI 78D3 CellsIn order to investigate whether the silkworm protein 30Kc6 had any protective effect 16574785 on HUVEC cells, which were incubated with Ox-LDL, silkworm protein 30Kc6 was expressed in BmN cells and was purified by Invitrogen Ni-NTA. It was then subjected to SDS-The Effect of the Silkworm Protein 30Kc6 on the JNK and p38 MAP Kinase ActivityThe MAP kinase family, MedChemExpress ITI007 especially the p38 and the JNK, have been reported to be involved in oxidative stress and cell apoptosis [10?2]. Therefore, the activities of p38 and JNK MAP kinase were examined by Western blot using antibodies (Fig. 7). The OxLDL significantly activated the p38 and the JNK MAP kinase inFunctional Analysis of Silkworm Protein 30KcFigure 3. Analysis of the purified 30Kc6 expressed in BmN cells by SDS-PAGE (A) and Western blot (B, C). Purified 30Kc6 proteins were transferred to nitrocellulose membrane after SDS-PAGE and analyzed by immuno-blotting with anti-66His-tag antibody and home-made polyclonal anti-30Kc6 antibody, respectively. M represents the protein molecular weight marker. 1. The purified silkworm fusion protein 30Kc6 in SDS-PAGE analysis; 2. The purified silkworm fusion protein 30Kc6 in Western blotting using the monoclonal anti-66His-tag antibody; 3. The purified silkworm fusion protein 30Kc6 in Western blotting using the home-made polyclonal anti-30Kc6 antibody. The arrows indicate the 30Kc6 protein. doi:10.1371/journal.pone.0068746.gHUVEC cells that were incubated with Ox-LDL. Furthermore, the 30Kc6 dramatically decreased the level of the activated p38 and JNK induced by Ox-LDL (Fig. 7), indicating that p38 and JNK MAP kinase were involved in the effect of 30Kc6 on HUVEC apoptosis. 30Kc6 might prevent the Ox-LDL-induced cell apoptosis by decreasing activation of the p38 and the JNK MAP kinase in the HUVEC cells.Protein Concentration Analysis of 30Kc6 in Freeze-dried Silkworm PupaProtein concentration of 30Kc6 in freeze-dried silkworm pupa meals was analyzed by ELISA. To set up the standard curvebetween the concentration of purified 30Kc6 sample and the OD value, the 30Kc6 proteins were diluted by double dilution with the initial concentration of 5 mg/mL. The OD492 value was measured by ELISA and the standard curve was constructed with the concentration of the 30Kc6 protein as the horizontal ordinate and the OD492 value as 23977191 the longitudinal coordinate. The linearregression analysis demonstrated that the constructed standard curve complied with regression equation (y = 0.3401x+0.0282, R2 = 0.9987). According to the equation, the concentration of 30Kc6 was 9.71 mg/g.Figure 4. Results of the DNA fragmentation (A) and the cell viability (B) in HUVEC.Ies and oxidative stress.Figure 2. Expression, purification (A) and polycnonal antibody generation (B) of 30Kc6 protein in E. coli. The 30Kc6 gene was inserted into prokaryotic expression plasmid pET-28a and was expressed in E. coli BL21 (DE3) with the induction of IPTG. M. represents the protein molecular weight marker. 1. Empty vector before inducing; 2. Empty vector after inducing; 3. Recombinant bacteria before inducing; 4. Recombinant bacteria after inducing (IPTG 0.1 mmol/L); 5. Recombinant bacteria after inducing (IPTG 1 mmol/L); 6. Purified 30Kc6 protein; 7. Purified antibody reacts with purified 30Kc6 protein; 8. Pre-immune rabbit serum reacts with purified 30Kc6 protein. The arrows indicate the 30Kc6 protein. doi:10.1371/journal.pone.0068746.gPurification and Identification of the Silkworm Protein 30Kc6 Expressed in BmN CellsIn order to investigate whether the silkworm protein 30Kc6 had any protective effect 16574785 on HUVEC cells, which were incubated with Ox-LDL, silkworm protein 30Kc6 was expressed in BmN cells and was purified by Invitrogen Ni-NTA. It was then subjected to SDS-The Effect of the Silkworm Protein 30Kc6 on the JNK and p38 MAP Kinase ActivityThe MAP kinase family, especially the p38 and the JNK, have been reported to be involved in oxidative stress and cell apoptosis [10?2]. Therefore, the activities of p38 and JNK MAP kinase were examined by Western blot using antibodies (Fig. 7). The OxLDL significantly activated the p38 and the JNK MAP kinase inFunctional Analysis of Silkworm Protein 30KcFigure 3. Analysis of the purified 30Kc6 expressed in BmN cells by SDS-PAGE (A) and Western blot (B, C). Purified 30Kc6 proteins were transferred to nitrocellulose membrane after SDS-PAGE and analyzed by immuno-blotting with anti-66His-tag antibody and home-made polyclonal anti-30Kc6 antibody, respectively. M represents the protein molecular weight marker. 1. The purified silkworm fusion protein 30Kc6 in SDS-PAGE analysis; 2. The purified silkworm fusion protein 30Kc6 in Western blotting using the monoclonal anti-66His-tag antibody; 3. The purified silkworm fusion protein 30Kc6 in Western blotting using the home-made polyclonal anti-30Kc6 antibody. The arrows indicate the 30Kc6 protein. doi:10.1371/journal.pone.0068746.gHUVEC cells that were incubated with Ox-LDL. Furthermore, the 30Kc6 dramatically decreased the level of the activated p38 and JNK induced by Ox-LDL (Fig. 7), indicating that p38 and JNK MAP kinase were involved in the effect of 30Kc6 on HUVEC apoptosis. 30Kc6 might prevent the Ox-LDL-induced cell apoptosis by decreasing activation of the p38 and the JNK MAP kinase in the HUVEC cells.Protein Concentration Analysis of 30Kc6 in Freeze-dried Silkworm PupaProtein concentration of 30Kc6 in freeze-dried silkworm pupa meals was analyzed by ELISA. To set up the standard curvebetween the concentration of purified 30Kc6 sample and the OD value, the 30Kc6 proteins were diluted by double dilution with the initial concentration of 5 mg/mL. The OD492 value was measured by ELISA and the standard curve was constructed with the concentration of the 30Kc6 protein as the horizontal ordinate and the OD492 value as 23977191 the longitudinal coordinate. The linearregression analysis demonstrated that the constructed standard curve complied with regression equation (y = 0.3401x+0.0282, R2 = 0.9987). According to the equation, the concentration of 30Kc6 was 9.71 mg/g.Figure 4. Results of the DNA fragmentation (A) and the cell viability (B) in HUVEC.
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