FIG 4.

VCP-UBXN1 is required for the turnover of BAG6 clients. (A) HEK-293T cells were transiently transfected with the indicated siRNAs and OPG-TASK. A duplicate set of samples derived from the same set of transfections was treated with bortezomib to demonstrate equal levels of transfection (bottom). Cell lysates were probed for OPG-TASK levels. (B and C) HEK-293T cells were transiently transfected with control, VCP (B), or UBXN1 (C) siRNAs and HA-PrP^mut. Levels of HA-PrP^mut were determined. (D) HEK-293T cells were transfected with HA-PrP^mut and then treated with cycloheximide in the presence or absence of the VCP inhibitor CB-5083 for the indicated times. Cells were lysed in 1% SDS, lysates were probed for the indicated proteins, and levels of the substrate were quantified by densitometry and normalized to PCNA levels. A.U, arbitrary units. (E and F) Wild-type (WT) and UBXN1 knockout (KO) HeLa Flp-in T-REX cells were transfected with HA-PrP^mut (E) or OPG-TASK (F) and treated with cycloheximide for the indicated times. Cells were lysed in 1% SDS, lysates were probed for the indicated proteins, and the levels of each substrate were quantified by densitometry and normalized to PCNA levels. (G) HEK-293T cells were transiently transfected with control, VCP, UBXN1, and BAG6 siRNAs, either alone or in combination, along with OPG-TASK. The levels of OPG-TASK were determined and quantified relative to control values. (H) HeLa Flp-in T-REX (wild-type and UBXD8 KO) cells were transfected with HA-PrP^mut, and the levels of PrP in lysates were determined. (I) HEK-293T cells were transfected with the indicated siRNAs and HA-PrP^mut. Cell lysates were probed with the indicated proteins. The levels of HA-PrP^mut were calculated by densitometry and normalized to PCNA levels (n = 3). n.s, not significant; *, P ≤ 0.05; **, P ≤ 0.01 (as determined by two-tailed Student's t test) (n = 3).