TLR9 Activation Is Triggered by the Excess of Stimulatory versus Inhibitory Motifs Present in Trypanosomatidae DNA
Figure 9
Analysis of DNA-HMGB1 complex by gel electrophoresis and Western blot.
L. major or vertebrate sonicated DNA (250 ng) were incubated with increasing amounts of HMGB1. Lane 1 shows the migration of DNA alone and lane H the HMGB1 alone. In lane 2 to 7, DNA was complexed to HMGB1, respectively at a molar ratio of 2.5, 5, 7.5, 15, 25 and 50. (A) Following electrophoretic migration samples were immediately blotted on a PVDF membrane and revealed with an anti-HMGB1 antibody. (B) Following electrophoretic migration, the gel was incubated in a solution of EtBr during 45 min to stain the DNA (left) then blotted and revealed with anti-HMGB1 antibody (right). (C) Sonicated Trypanosomatidae DNA alone (250 ng) (lane 1) or complexed with HMGB1 at a molar ratio of 25 and 50 (lanes 6 and 7) were analysed by electrophoresis with the procedure described in B. Free HMGB1 (25 kDa) did not migrate in the 1% agarose gel.