Figure 2
From: Lysine 222 in PPAR γ1 functions as the key site of MuRF2-mediated ubiquitination modification
Identification of ubiquitination site(s) in vitro by GST-pulldown assay (original blots are presented in Supplementary Figs. S1–S3). (a) The upper: the over-expression plasmid of PPAR γ1 or the mutant of PPAR γ1 (K68R, K222R, K228R, K242R, K356R) was transfected in HEK293T cells respectively. The PPAR γ1 proteins levels were verified by immunoblot, and no endogenous PPAR γ1 was observed in 293T cells. The lower: immunoblot analysis of the purification efficiency of PPAR γ1 protein. Compared to the negative and positive controls, no GAPDH protein was detected in the total protein derived from the PPAR γ1 and mutants transfected groups, indicating that purified proteins of PPAR γ1 and the mutants were obtained. (b) The upper: MuRF2 auto-ubiquitination was demonstrated by immunoblot of MuRF2 (lane 5 and lane 6). The lower: MuRF2’s ability to ubiquitinate PPAR γ1. The smeared PPAR γ1 was observed obviously in the full reaction (the far right lane 6). (c) Residue K68 showed the remote possibility of being the ubiquitination site. Except the PPAR γ1 K68R, all the protein stability of the mutant PPAR γ1 K222, K228, and K242 and K356 were weakened in the presence of MuRF2 compared to that of the PPAR γ1 protein, indicating the dispensability of lysine site K68 in MuRF2 mediated PPAR γ1 ubiquitination modification. Ub, ubiquitin; E1, ubiquitin activating enzyme; E2, ubiquitin conjugating enzyme.
