TRIM32 facilitates the accumulation of K63 ubiquitin chain-containing Glis2. Glis2 was co-expressed with HA-tagged, wild-type ubiquitin (Ubi-WT), HA-tagged ubiquitin K48R (Ubi-K48R), or HA-tagged ubiquitin K63R (Ubi-K63R) in HEK 293T cells. In A, cells were incubated for the last 2 h with 12 μm MG132. In B, cells were co-transfected with V5-tagged TRIM32 (V5.TRIM32). K63-linked ubiquitin chain accumulated in the presence of V5.TRIM32, but not after incubation with MG132, using a K63 specific antibody. IP, immunoprecipitation; WB, Western blotting.