FIG 4.

p97 binds to ubiquitinated IκBα. An IP-Western blotting approach was employed to examine the association of p97 with ubiquitinated IκBα. (A) Flag/HA-p97 was expressed in HeLa cells. (B) Flag/HA-p97 was immunoprecipitated from HeLa-Flag/HA-p97 cells using anti-HA antibody. The association of p97 with ubiquitinated IκBα was verified using anti-IκBα antibody. (C) HeLa and HeLa-Flag/HA-p97 cells were treated with MG132 for 30 min and then with TNF-α for 10 min or no further treatment. Cells were lysed for IP with anti-HA antibody, followed by Western blotting using anti-IκBα antibody. (D) Endogenous IκBα was immunoprecipitated using a polyclonal anti-IκBα antibody and confirmed with anti-IκBα antibody. (E) The interaction between endogenous IκBα and p97 was confirmed by Western blotting using anti-p97 antibody.