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L502P and C833A UBE3A mutants increase S5a ubiquitination. HEK293 cells were transfected with HA-tagged ubiquitin, S5a, UBE3A wild-type, C833A or L502P, alone or in combination. After 24 h, cells were collected and complexes were immunoprecipitated with anti-HA-conjugated agarose beads. Complexes were then analyzed by western blotting for S5a using anti-S5a antibody. Inputs (10%) for S5a and UBE3A are also shown
