Figure 1. Modification of vimentin by various electrophiles.

(a) Structures of the electrophilic lipids used. Asterisks denote carbons susceptible of Michael addition. (b) Vimentin was incubated with vehicle, PGA₁-B, 15d-PGJ₂-B or Iac-B at 10 μM, or 50 μM, HNE for 2 h at 37 °C. Incorporation of electrophiles as well as vimentin levels were assessed by WB. The position of molecular weight (MW) standards (in kDa) is indicated on the left of the panels. Dividing lines indicate where lanes from the same gel have been cropped. (c) Competition between various electrophiles for binding to vimentin. Vimentin was preincubated with 100 μM HNE for 2 h at 37 °C, after which, 10 μM Iac-B or 1 μM PGA₁-B were added and incubation was continued for 30 min at r.t. (d) Effect of vimentin modification by electrophiles on polymerization. Vimentin was incubated with vehicle (control), 50 μM HNE (upper panel) or 100 μM Iac-B (lower panel) for 2 h at 37 °C, polymerized with 150 mM NaCl, and soluble (S) and polymerized (P) vimentin were separated by ultracentrifugation. Incorporation of Iac-B into vimentin was assessed as above. (e) Electron microscopy (EM) of vimentin filaments formed before or after treatment with HNE. Vimentin was incubated in the absence or presence of HNE as in d, before polymerization with 150 mM NaCl and visualization by EM. Scale bar, 500 nm. (f) Incorporation of PGA₁-B into soluble or polymerized vimentin. Soluble (ct) or polymerized (pol) vimentin was incubated with 10 μM PGA₁-B and its binding to vimentin was assessed as above; n≥3 for all blots. (g) Effect of various electrophiles on the vimentin cytoskeleton in RMC. RMCs were transfected with GFP-vimentin wt or C328S and treated 48 h later with 7.5 μM 15d-PGJ₂, 30 μM PGA₁ or 10 μM HNE for 4 h and observed live by confocal microscopy. Scale bars, 20 μm. The proportion of cells with condensed vimentin was estimated, right panel (mean±s.e.m. of three independent experiments, *P<0.05 by Student's t-test). (h) RMC treated with 15d-PGJ₂ or PGA₁ for 22 h or with HNE for 4 h were lysed and vimentin levels assessed by WB.