Figure 6. Knocking down WWP1 reduces LPS–induced K48-linked, but not K63-linked, polyubiquitination of endogenous TRAF6.

(A) Peritoneal macrophages (2×10⁶) were infected with the Lesh NC and Lesh WWP1 virus particles (m.o.i 40) for 48 h, and then treated with LPS (at a final concentration of 400 ng/mL) for the indicated times. Whole cell lysates were subjected to immunoprecipitation with anti-TRAF6, followed by Western blot analysis with anti-Ub, anti-Lys48 specific Ub and anti-Lys63 specific Ub. Cell lysates were also analyzed by immunoblotting with anti-TRAF6 or anti-WWP1. Data are from one experiment representative of three independent experiments with similar results. (B) The RAW264.7 stable cell lines of WWP1 and C890A (4×10⁵) were stimulated by LPS (at a final concentration of 400 ng/mL) for the indicated times. Whole cell lysates were subjected to immunoprecipitation with anti-TRAF6 followed by Western blot analysis with anti-Ub, anti-Lys48 specific Ub and anti-Lys63 specific Ub. Cell lysates were also analyzed by immunoblotting with anti-TRAF6 or anti-WWP1. Data are from one experiment representative of three independent experiments with similar results.