PR39 does not affect known steps in IκBα processing. (a) The effect of PR39 on IκBα phosphorylation in cell culture. U937 cells cultured in the absence (–) or presence (+) of pretreatment with 10 μM PR39 were exposed to 1 ng/mL TNF-α; 45 minutes later the cells were lysed and subjected to SDS-PAGE and Western blotting with anti-IκBα Ab. Note increased amount of the typical-appearing phosphorylated IκBα band in PR39-treated cells. (b) The effect of PR39 on IκBα ubiquitination in vitro. In vitro ubiquitination of phosphorylated IκBα protein was carried out using crude HeLa-cell extract in the presence of control peptides (lanes 3 and 4), PR39 (lanes 1 and 2), or in the absence of both (lane 5). Note that the addition of either PR39 at two different concentrations or of a control (random) peptide failed to affect IκBα ubiquitination. At the same time, no ubiquitination of phosphorylation site IκBα mutants (lanes 6 and 7) or unphosphorylated IκBα (weight IκBα, lane 8) was detected. (c) The effect of PR39 on IκBα ubiquitination in cell culture. HA-Ub–expressing ECV304 cells were exposed to 1 ng/mL TNF-α in the presence (+) or absence (–) of 10 μM PR39 pretreatment; 45 minutes later the cells were lysed and subjected to IκBα immunoprecipitation followed by Western blotting of the pellet material with anti-HA antibody. Note multiple ubiquitinated IκBα intermediaries in PR39-treated, but not control cells. (d) PR39 does not inhibit IκBα-VCP binding. Wild-type ECV304 cells treated as described in c were subjected to immunoprecipitation with anti-IκBα antibody (IκBα-IP) followed by Western blotting with the anti–VCP-3 Ab. Note the presence of 90-kD VCP band in the presence or absence of PR39 treatment.