Phosphorylation of unexpanded polyQ Httex1p and 586aa Htt is associated with its reduced abundance in cell culture. (A) Levels of unexpanded polyQ Httex1p are reduced with overexpression of IKK-β; this effect is inhibited with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4 was cotransfected with myc-actin and with vector or IKK-β into St14A cells. Cells were treated for 16 h with DMSO, 100 nM epoxomicin in DMSO, or 20 mM ammonium chloride/100 µM leupeptin in DMSO. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc to detect myc-actin, and anti-HA to detect Httex1p. (B) IKK-β overexpression reduces levels of unexpanded polyQ Httex1p in the presence of proteasome or lysosome inhibition. Scion software was used to quantitate triplicate levels of 25QP-H4 from the experiment represented in A, normalized to levels of myc-actin transfection control, within each treatment group: control, epoxomicin, or ammonium chloride/leupeptin. (C) Mimicking phosphorylation of unexpanded polyQ Httex1p reduces its abundance in cell culture; this effect is reduced with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4, wt control or QEE, EE, AA, or 3R were cotransfected with myc-actin into St14A cells. Cells and lysates were treated as in A. (D) Levels of phosphorylated unexpanded polyQ 586aa Htt accumulate with inhibition of the proteasome or the lysosome; phosphorylation is reduced with expansion of the polyQ repeat. 15Q or 128Q 586aa Htt constructs were cotransfected into St14A cells with myc-actin and with vector or IKK-β. Cells were treated for 4 h with DMSO or 100 nM epoxomicin in DMSO (to eliminate any possible effect on the lysosome by epoxomicin), or for 16 h with water or 20 mM ammonium chloride/100 µM leupeptin in water. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc, anti–S13-P, and anti-Htt 3B5H10.