Figure 4
From: Lysine 222 in PPAR γ1 functions as the key site of MuRF2-mediated ubiquitination modification
Confirmation of K222 is the key site of MuRF2 ubiquitination modification PPAR γ1 (original blot is presented in Supplementary Fig. S6). (a) Function of K222 in PPAR γ1 stability. HEK 293T cells were co-transfected with plasmids His-MuRF2, HA-Ub and PPAR γ1 or K222R mutant. The cells were treated with CHX (final concentration 60 μg/mL) for 3 h and 6 h respectively before harvest. The proteins turnover of PPAR γ1 and PPAR γ1 K222R were determined by immunoblot. Signals of the PPAR γ1 from immunoblots were analyzed using the Image J (National Institutes of Health) and normalized by GAPDH signal. (b) Percentage of the remaining PPAR γ1 and K222R proteins. Compared to the significantly weakened PPAR γ1 protein, the remaining K222R proteins remained unchanged after 3 h and 6 h of CHX administration (p < 0.05). (c) HEK 293 T cells were transfected with plasmids of His-MuRF2, HA-Ub and PPAR γ1 or the K222R mutant. Samples were subjected to quantitative-PCR analysis of cardiac genes ACOX1, PLIN2 and CPT1b. Data were presented as mean ± SD. n = 3. #p < 0.05 by two-tailed Student’s t-test. (d) The 3D spatial structure of PPAR γ1 ligating ubiquitin derived by PyMOL software (version 2.4.0 openvr; DeLano Scientific, San Carlos, CA, USA; https://pymol.org/2/). At position 76 of glycine carboxyl, ubiquitin ligates PPAR γ1 at lysine 222 site. The green is ubiquitin and the blue is PPAR γ1.
