Journal: PLoS pathogens

Article Title: Exploitation of the complement system by oncogenic Kaposi's sarcoma-associated herpesvirus for cell survival and persistent infection.

doi: 10.1371/journal.ppat.1004412

Figure Lengend Snippet: Figure 7. Activation of complement pathway induces STAT3 phosphorylation to enhance cell survival of latently KSHV-infected endothelial cells. (A) The enhanced cell survival of KSHV-infected endothelial cells by complement was mediated by the STAT3 pathway. TIME- KSHV cells were cultured for 48 h in normal human serum in growth factor-depleted medium with and without JAK or STAT3 inhibitor, and the numbers of dead cells (left panel) and live cells (right panel) were determined. Cells cultured in heat-inactivated human serum were used as controls. Results are means 6 SD from three independent experiments with three repeats. * P,0.05, ** P,0.01 and *** P,0.001 by Student’s t-test. (B) Complement activation induced STAT3 tyrosine phosphorylation in TIME-KSHV cells. STAT3 tyrosine (Y705) and serine (S727) phosphorylation in TIME and TIME-KSHV cells switched from full endothelial cell medium with growth factors and 10% heat-inactivated human serum to endothelial cell medium depleted of growth factors with 10% heat-inactivated or normal human serum for the specified lengths of time. (C) Complement activation was required for the enhanced STAT3 tyrosine phosphorylation of TIME-KSHV cells. STAT3 tyrosine phosphorylation was examined in cells cultured for 24 h in 10% C3-depleted human serum or C3-depleted human serum reconstituted with purified C3 protein in endothelial cell medium depleted of growth factors. (D) Formation of C5b-9 complexes was required for the enhanced STAT3 tyrosine phosphorylation. STAT3 tyrosine phosphorylation was examined in cells cultured for 24 h in 10% C6-depleted human serum or C6-depleted human serum reconstituted with purified C6 protein in endothelial cell medium deprived of growth factors. (E) STAT3 tyrosine phosphorylation was unchanged without the continuous presence of normal human serum and growth factors. STAT3 tyrosine phosphorylation was examined in cells cultured in 10% heat-inactivated or normal human serum for 1 h, and then in endothelial cell medium without any serum and growth factors for additional 23 h. (F) JAK but not Src activation mediated STAT3 tyrosine phosphorylation. STAT3 tyrosine phosphorylations was examined in TIME-KSHV cells cultured in 10% normal human serum in endothelial cell medium depleted of growth factors for 24 h with or without the presence of STAT3, JAK or Src inhibitor. doi:10.1371/journal.ppat.1004412.g007

Article Snippet: C1q-depleted human serum, C3-depleted human serum, C6-depleted human serum, factor B-depleted human serum, purified C3 protein, and purified C6 protein were purchased from Quidel Corporation (San Diego, CA).

Techniques: Activation Assay, Phospho-proteomics, Infection, Cell Culture, Purification

Journal: PLoS ONE

Article Title: Cleavage of Kininogen and Subsequent Bradykinin Release by the Complement Component: Mannose-Binding Lectin-Associated Serine Protease (MASP)-1

doi: 10.1371/journal.pone.0020036

Figure Lengend Snippet: Prekallikrein (100 µg/ml) was incubated in HBS buffer (see ) at 37°C (time in minutes indicated) in the presence of 5 µg/ml kallikrein or rMASP-1. 10 µl samples per lane were analyzed by reducing SDS-PAGE. The contrast was somewhat enhanced for the better visibility of faint bands.

Article Snippet: Human plasma kallikrein (referred to as kallikrein) and human plasma prekallikrein (referred to as prekallikrein) were from Innovative Research (Novi, MI).

Techniques: Incubation, SDS Page