Journal: Molecular medicine (Cambridge, Mass.)

Article Title: The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88.

doi: 10.2119/molmed.2015.00197

Figure Lengend Snippet: Figure 3. SQV inhibits the interaction between cathepsin V and the TLR4-Myddosome recep- tor signaling complex. (A,B) HEK293 cells transfected with HA tagged TLR4 were immunopre- cipitated for HA:TLR4 or cathepsin V followed by subsequent cathepsin V and or TLR4:HA im- munobloting. (C) Recombinant human TLR4 and cathepsin V binding was measured by surface plasmon resonance. SQV at 0.625, 1.25, 2.5, 5 μmol/L dose dependently inhibited binding of TLR4 to cathepsin V. (D) Human monocyte-derived macrophages were pre- treated with SQV (30 μmol/L for 1h) followed by the addition of HMGB1 for 30 and 60 min. Representative immunoblot of immunoprecipitated cathepsin V or MYD88 blotted against TLR4 and IRAK4. (E) Representative immunoblot of TLR4, IRAK4, cathepsin V and MYD88 total protein expression used as input for the experiments in (D). (F) Histogram of quantitation of multiple experiments from (D). (G) Effect of SQV on HMGB1-induced IRAK-1 protein expres- sion compared with β-actin as determined by Western blot analysis.

Article Snippet: Anti-human cathepsin V (MAB10801, R&D Systems), anti-HA (ab-hatag, InvivoGen), anti-human TLR4 (IMG-578A, IMGENEX), anti-human MyD88 (4283, Cell Signaling), anti-human IRAK4 (4363, Cell Signaling ), anti-human IRAK1 (4504, Cell Signaling).

Techniques: Transfection, Recombinant, Binding Assay, SPR Assay, Derivative Assay, Western Blot, Immunoprecipitation, Expressing, Quantitation Assay