NOSIP monoubiquitinates the catalytic subunit of PP2A.

<p>(A) <i>In vitro</i> ubiquitination assay. An assay without E1 enzyme served as negative control. Ubiquitinated PP2A is detected with α-PP2Ac and α-Flag (Flag-tag of ubiquitin), IB with α-NOSIP served as control. Arrowheads indicate monoubiquitinated PP2Ac; * heavy chain of the antibody; ** originates from the commercial Flag-ubiquitin; the upper bands detected in both lanes are considered unspecific reactivity of the Flag-antibody. (B) <i>In vivo</i> ubiquitination assay with rescue of PP2Ac monoubiquitination by the expression of NOSIP. MEFs of the indicated genotype were infected with Flag-ubiquitin (Flag-ubi) plus NOSIP or empty vector (control). Immunoprecipitation (IP) was performed with α-PP2Ac and proteins were detected by IB with α-Flag (Flag-tag of ubiquitin). IB using α-PP2Ac, α-NOSIP and α-vinculin served as controls. Arrowhead indicates monoubiquitinated PP2Ac; * heavy chain of the antibody (C) <i>In vivo</i> ubiquitination assay with proteasomal inhibition. WT MEFs were infected with Flag-ubiquitin (Flag-ubi) and either left untreated (−) or pre-treated with 20 µM MG-132 for 5 h (MG) and monoubiquitinated PP2Ac was detected as described above. IB using α-ubiquitin served as control for proteasome inhibition. Arrowhead indicates monoubiquitinated PP2Ac; * heavy chain of the antibody. (D) Protein levels of different PP2A subunits in MEFs from WT, HET and NOSIP KO mice were analysed by IB with PP2A subunit-specific antibodies and α-NOSIP. IB using α-GAPDH is shown as loading control. IB using α-ubiquitin served as control for proteasome inhibition. MEFs were left untreated (−), treated with 20 µM MG-132 for 5 h (MG) or treated with 100 nM okadaic acid for 3 h (OA).