Figure 7
The binding capability of PLTP to LPS and the macrophage toxicity determination of PLTP-LPS complex.
For co-incubation assay, active recombinant PLTP (rPLTP), rPLTP treated in 56 °C for 30 min (56-rPLTP) and rPLTP treated in 95 °C for 5 min (95-rPLTP) were incubated with LPS in a final volume of 50 μL for 60 min, respectively. The mixture was loaded in 10% native gel for electrophoresis, staining, immunoblot and photographing. (See Materials and Methods for detail) (A,B) the levels of TNFα and IL-6 in media of PLTP−/− BMDM after the stimulation of rPLTP-LPS mixture or albumin-LPS mixture for 24 h, respectively. (C,D) the levels of TNFα and IL-6 in media in medium of WT BMDM after the stimulation of rPLTP-LPS mixture or albumin-LPS mixture for 24 h, respectively. (E) The rPLTP, 95-rPLTP, albumin and 56-rPLTP were incubated with LPS, respectively. The mixtures were run in native gel and photographed after Coomassie blue staining. (F) The rPLTP, albumin and 95-rPLTP were incubated with LPS, respectively. The mixture was run in native gel and photographed after Coomassie blue staining. (G) FITC conjugated LPS (FITC-LPS) was incubated with rPLTP and albumin, respectively. The mixtures were run in native gel and the fluorescence was photographed (Excitation: 488 nm, Emission: 525 nm). The full length gel was in supplementary-figure-2; (H) FITC-LPS was incubated with rPLTP, 56-rPLTP, 95-rPLTP, respectively. Purchased recombinant PLTP (pPLTP) and pPLTP treated in 95 °C for 5 min (95-pPLTP) were loaded as positive control and negative control for LPS incubation, fluorescence photographing and WB analysis, respectively. Left panel, the WB results of co-incubated mixture, detected by PLTP antibody; intermediate panel, FITC signals detected by fluorescence photographing; right panel, the merge picture. The assays were conducted for 3 times and the whole length gels or blots were showed. (I) rPLTP/LPS mixture was co-incubated with RAW264.7 cells for 24 h and the cell viability was assayed by MTT. Data are expressed as the mean ± SD (n = 4 for experiments in (A–D) n = 3 for experiments in (E–H) n = 6 for experiments in (I)). *P < 0.05; **P < 0.05; ***P < 0.05.
