(A) Growth assay as in Figure 1A of HeLa-µs cells, in which HRD1 was deleted (KO), SEL1L was silenced (KD), or not (WT). Cells were treated with 0.5 nM mifepristone (Mif) to induce expression of µs (+), or not (-), and WT cells were treated with Kif or not (ctrl), as indicated. (B,C) Immunoblots of µs harvested from WT, HRD1 KO (B,C), or SEL1L KD (C) HeLa-µs cells that were induced with 0.5 nM Mif to express µs for 4 hr and then treated for the indicated times with 100 μg/ml CHX either alone (B,C), in combination with 20 mM Kif, 100 nM BafA1, or not (ctrl) (B), or 10 µg/ml MG132 (C), as indicated. The arrowhead indicates the deglycosylated form of µs. (D) HeLa-µs WT or HRD1 KO cells were induced with Mif (0.5 nM) for 24 hr to express µs or not (ctrl) , as indicated. Samples were lysed in 1% lauryl maltose neopentyl glycol (LMNG) and sedimented over a 10–40% sucrose gradient. Levels of µs, HRD1, SEL1L, Derlin-2, and OS-9 were detected by immunoblotting. Note that in HRD1 KO cells leaky expression of µs becomes apparent due to the lack of ERAD. At low expression levels, however, µs does not form high molecular weight aggregates, indicative of the adequacy of the chaperoning machinery.