FIG. 2.

Serine 932 is essential for TNF-α to trigger proteolysis of p105. (A) Clones of HeLa cells stably transfected with HA-p105, HA-p105(S923A), HA-p105(S927A), or HA-p105(S932A) were metabolically pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine (45 min) and then chased for the indicated times in complete medium (control) or complete medium supplemented with TNF-α. Anti-HA immunoprecipitates were resolved by SDS-7.5% PAGE and revealed by fluorography. Amounts of immunoprecipitated wild-type (WT) and point-mutated HA-p105 were quantified by laser densitometry (mean ± standard error of the mean; n = 5). (B) HeLa cells stably transfected with either HA-p105 or HA-p105(S932A) were analyzed by pulse-chase metabolic labeling as described for panel A. Anti-HA immunoprecipitates (Ip) were resolved by SDS-7.5% PAGE and revealed by fluorography. The position of HA-p105 is indicated with an arrow. (C) Cell lysates from the indicated HeLa clones with or without TNF-α stimulation (15 min) were Western blotted for endogenous IκBα. (D) Stably transfected HeLa cells were preincubated for 1 h with LLnL proteasome inhibitor and then stimulated for 15 min with TNF-α or control medium. Immunoprecipitates of wild-type and point-mutated HA-p105 were then sequentially Western blotted with the indicated antibodies.