Figure 4: Lys28 is required for PPARγ-mediated p65 degradation.

(a) HEK293T cells were transfected with GST-p65 and PPARγ, and cell lysates were subjected to GST pull-down and SDS–PAGE. The Coomassie-stained proteins were excised and subjected to LC/MS/MS analysis. One of seven lysine residues of ubiquitin produces an isopeptide linkage with the C-terminus of another ubiqutin moiety, forming ubiquitin chains of various lengths and shapes (Lys 6, Lys 11, Lys 27, Lys 29, Lys 33 and Lys 63), but trypsin cannot cleave Gly–Gly modified lysine, therefore, ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin by trypsin cleavage. The full tryptic peptide LIFAGK*QLEDGR with K48 modified by GG is shown. K* depicts the lysine residue modified by isopeptide linkages. (b) HEK293T cells were transfected with HA-PPARγ, HA-ubiquitin (Ub) or HA-ubiquitin mutant (K0 or K48R) plasmids. Cell lysates were subjected to denatured immunoprecipitation and western blotting. Cells were treated with 10 μM MG132 for 6 h before cell lysis. (c) A schematic representation of the lysine mutants of p65/RHD used in this study (upper panel). (d) HEK293T cells were transfected with HA-PPARγ, GST-p65 or with the K→R mutant plasmids. Cell lysates were subjected to western blotting. (e) NFκB promoter activity in HA-PPARγ, p65 or the K→R mutants transfected in HEK293T cells. Results are expressed as means±s.e.m. (n=3).