Immunoblot Analysis of Transcription Factors

Total proteins that were extracted from EL4 cells with RIPA buffer containing phosphatase inhibitor as well as protease inhibitor were separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and were subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore). After PVDF membrane was blocked with 5% nonfat milk for 2 h, it was sequentially incubated overnight at 4 ℃ with the following primary antibodies: PPARγ (CST, Danvers, MA, USA), ELK1 (CST), ETS1 (Proteintech, Rosemont, USA), Foxp3 (Abcam, Cambridge, UK), and Sp1 (Abcam). Horseradish peroxidase (HRP)-labeled goat anti-mouse antibody (Proteintech) or HRP-labeled goat anti-rabbit antibody (Proteintech) was utilized as the secondary antibody [22 (link)]. The proteins were subsequently revealed with enhanced chemiluminescence (ECL, Meilunbio, Dalian, China). The density of the bands (versus GAPDH) was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).