In the article titled “Pioglitazone Attenuates Drug-Eluting Stent-Induced Proinflammatory State in Patients by Blocking Ubiquitination of PPAR” [1], there are additional methods that should be added to the article which are mainly about the methods for PPAR gamma binding and ubiquitination. The additional methods are shown below:
2.8. Immunoblotting. Protein levels of p65, p50, PPAR-γ, and Gapdh of MNC were detected by western blotting with Santa Cruz antibodies against the p65 subunit (sc-372), p50 subunit (sc-114), PPAR-γ (sc-7273), and Gapdh (SC-48166) as described previously . Mononuclear cells were lysed in RIPA buffer (Cybrdi, Gaithersburg, MD, USA) that contained protease inhibitor (Roche Diagnostics, Indianapolis, IN, USA). Equal amounts of protein were loaded onto 4–12% Bis-Tris precast gels for electrophoresis and were electrotransferred onto a PVDF membrane (Roche Diagnostics). After blocking for 1 h at room temperature, membranes were sequentially incubated with primary Abs at 4°C overnight and secondary Abs at room temperature for 1 h. The protein signal was detected by chemiluminescence and all values were corrected by loading with Gapdh.
2.9. Immunoprecipitation. Immunoprecipitation assays were performed as described previously . Cell extracts were prepared by using modified RIPA buffer (Cell Signaling Technology). Cell lysates (300 μg) were incubated with 1 μg of PPAR Ab (sc-7273) at 4°C overnight and precipitated with protein G agarose beads (Santa Cruz) at 4°C for 2 h. Immunoprecipitated proteins were separated by SDS-PAGE and transferred onto a PVDF membrane. Membranes were then sequentially incubated with primary ubiquitination Ab (SC-8017) and secondary Abs. Bands were visualized by using an ECL system.
In addition, the below part should be added to the Discussion section after the third paragraph:
“The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors which regulate adipocyte differentiation, glucose homeostasis, and macrophage activation . PPAR-γ ubiquitination and degradation have been found in adipocyte, and proteasome inhibitors inhibited the downregulation of PPAR-γ. Proinflammation cytokine was also found to induce PPAR-γ degradation . Herein, we found a novel mechanism such that PIO enhances PPAR-γ binding activity though inhibiting its ubiquitination and degradation, which may be important for TZDs clinical use.”
Moreover, the references below should be included in the References list: T. Chen, X. Jin, B. H. Crawford et al., “Cardioprotection from oxidative stress in the newborn heart by activation of PPARg is mediated by catalase,” Free Radical Biology & Medicine, vol. 53, no. 2, pp. 208–215, 2012. C. Xie, T. Yagai, Y. Luo et al., “Activation of intestinal hypoxia-inducible factor 2α during obesity contributes to hepatic steatosis,” Nature Medicine, vol. 23, no. 11, pp. 1298–1308, 2017. Q. Zhao, D. Zhou, H. You et al., “IFN-γ aggravates neointimal hyperplasia by inducing endoplasmic reticulum stress and apoptosis in macrophages by promoting ubiquitin-dependent liver X receptor-α degradation,” The FASEB Journal, vol. 31, no. 12, pp. 5321-5331, 2017. L. Fajas, D. Auboeuf, E. Raspé et al., “The organization, promoter analysis, and expression of the human PPARgamma gene,” The Journal of Biological Chemistry, vol. 272, no. 30, pp.18779–89, 1997. S. Hauser, G. Adelmant, P. Sarraf, H. M. Wright, E. Mueller, B. M. Spiegelman, “Degradation of the peroxisome proliferator-activated receptor gamma is linked to ligand-dependent activation,” The Journal of Biological Chemistry, vol. 275, no. 24, pp. 18527–18533, 2000. K. J. Waite, Z. E. Floyd, P. Arbour-Reily, J. M. Stephens, “Interferon-gamma-induced regulation of peroxisome proliferator-activated receptor gamma and STATs in adipocytes,” The Journal of Biological Chemistry, vol. 276, no. 10, pp. 7062–7068, 2001.