Fig. 3.

IRF3 is activated by TLR3 or RLH stimulation. a Human podocytes were left unstimulated (-) or stimulated by Poly(I:C) transfection [RLH, 2 µg/ml Poly(I:C) + 3 µl/ml FuGENE6] or by adding 50 µg/ml Poly(I:C) to the culture medium (TLR3) for 8 h. The cell lysates were probed by Western blot for human P56 and P60. b Podocytes from TLR3−/− mice were left unstimulated (-) or treated by adding (TLR3) or transfecting (RLH) Poly(I:C); after 8 h, cell lysates were tested for mouse P54 by Western blot. c Human podocytes were stimulated by adding varying concentrations (0–100 µg/ml) of Poly(I:C) as indicated for 8 h; blots from cell lysates were probed for P56 or P60. d Human podocytes were stimulated by adding 50 µg/ml Poly(I:C) for varying incubation times (0–24 h), as indicated; blots were stained for P56 or P60. Immunoblotting for actin served as a loading control for all Western blots. e Human podocytes, grown on coverslips, were left unstimulated (-) or stimulated by transfection with (RLH) or addition of (TLR3) Poly(I:C) for 3 h, as above. Cells were then stained with anti-IRF3 Ab. f Mouse podocytes were grown on coverslips and left untreated (-) or transfected (RLH) or added (TLR3) Poly(I:C) for 3 h, as above and stained with anti-IRF3 Ab. g Cytosolic (CP) or nuclear (Nuc) proteins fractionated from human podocytes were collected 3 h after no addition of dsRNA (-) or after RLH or TLR3 stimulation. IRF3 in the fractions was detected by Western blot. The membranes were also probed for DRBP76 and tubulin as markers for nuclear and cytoplasmic proteins, respectively. h Phosphorylation of IRF3 at serine 396 (pS³⁹⁶ IRF3) was tested by Western blots applied to lysates from human podocytes left untreated or stimulated for 3 h by RLH or TLR3 ligands (as above). Immunostains for total IRF3 and actin were used as loading controls.