Figure 6

TRIM32 ubiquitinates recognized malin substrates such as R5/PTG. A) HEK293 cells were co-transfected with 6xHis-tagged ubiquitin constructs, myc-R5/PTG, and either pCINEO-TRIM32, pCINEO-TRIM32 (H42A) (encoding a catalytically inactive form of TRIM32) or empty vector. Cell extracts were obtained as described in Materials and Methods, and samples from crude extracts (CE, 30 μg) and material bound to the metal affinity column (Bound) were analyzed by SDS-PAGE and Western blotting using anti-myc antibodies, as described in Materials and Methods. B) MEF cells from epm2b-/- mice lacking malin were co-transfected with 6xHis-tagged ubiquitin constructs, myc-R5/PTG and pFLAG-TRIM32 or empty plasmids. Cell extracts were analyzed as in part A.