Figure 4.
Phosphorylated and acetylated endogenous wt Htt accumulates with inhibition of the proteasome and lysosome. (A) Overexpression of IKK-β increases phosphorylation of endogenous Htt and IκBα in cell culture. St14A cells were transiently transfected with vector, IKK-β, or IKK-α, together with vector or two different pools of anti–IKK-αβ shRNAs; a control pool that did not silence IKK well (pool 1) and one pool that was effective in silencing IKK (pool 2), as assessed by levels of FLAG-IKK-α and -β by Western. Lysates were subjected to Western analysis with anti-Htt MAB2166, anti–S13-P, anti–K9-Ac, anti-phosphoserine 32 IκBα (IκBα-P), anti–IκBα, anti–α-tubulin, and anti-FLAG to detect FLAG-tagged IKK-β and IKK-α. Bands the size of full-length endogenous Htt (350 kD) are shown by the arrow on the left. Boxed bands show the reduction in phosphorylated IκBα with IKK shRNA. (B) Phosphorylated and acetylated Htt accumulate with inhibition of the proteasome or the lysosome. St14A cells were transiently transfected with IKK-β or vector, and were treated as in Fig. 3 D. Lysates were subjected to Western analysis with anti–α-tubulin, and anti-Htt antibodies MAB2166, anti–S13-P or anti–K9-Ac. (C) Pharmacological activation of the IKK complex modulates levels of phosphorylated Htt and IκBα. St14A cells were incubated with 20 ng/ml TNF-α or IL-1β over a time course. Lysates were subjected to Western analysis as in A with additional detection by anti-Htt Ab1. (D) IL-1β–induced Htt S13-phosphorylated species accumulate with inhibition of the proteasome or lysosome. St14A cells were treated for 2 h with 20 ng/ml Il-1β before lysis. The proteasome and lysosome were inhibited, and Western analysis performed as in B.