Role of dimerization in SULT4A1 stability.
(A) IMR-32 cells transfected with wild-type (WT), TV>AE mutant or KTV>QAE mutant SULT4A1 were treated with 10 µg/ml cycloheximide and protein was collected at the different times. Western blots of protein were quantified by densitometry and normalized to tubulin as a loading control. Data are expressed relative to protein levels at time 0 (% Control). All results are mean ± s.e.m, n = 3. (B) Polyubiquitination of SULT4A1 wild-type and mutant proteins. IMR-32 cells were transfected with the different SULT4A1 FLAG-tagged constructs along with a HA-tagged ubiquitin construct. Following treatment with 20 µM MG132 for 8 hr, Western blots of HA-tagged proteins were performed. Expression of each construct was confirmed with anti-FLAG antibody (Input). Left panel represents immunopreciptation before protein denaturation while the right panel represents immunoprecipitation after protein denaturation. (C) Western blot for HA-ubiquitin on supernatants prior to immunoprecipitation.