The laforin-malin complex does not ubiquitinate TRIM32-related substrates such as dysbindin and PIASy. A) Ubiquitination of dysbindin. HEK293 cells were transfected with 6xHis-tagged ubiquitin construct, pmyc-dysbindin and plasmids encoding TRIM32 [wild type or a catalytically inactive form (H42A)] or plasmids encoding laforin and malin (pCMV-HA-laforin and pCDNA3-HA-malin). Samples were processed and analyzed as in Figure 6. Crude extracts were also analyzed for the presence of laforin and malin using an anti-HA antibody. B) Ubiquitination of PIASy. Similar experiments as those performed in part A) were conducted in cells expressing myc-PIASy. In addition we probed the action of Mdm2, another E3-ubiquitin ligase. Crude extracts were analyzed for the presence of laforin and malin using an anti-HA antibody, and with an anti-Mdm2.
Romá-Mateo et al. BMC Evolutionary Biology 2011 11:225 doi:10.1186/1471-2148-11-225