2.6 | Western blot
Aortas or SMCs cultured in 0.2% FBS DMEM overnight were lysed in RIPA buffer (Enogene, Cat# E1WP106). Experimental details of Western blots are in accordance with BJP guidelines (Alexander et al., 2018). In some experiments, protein was extracted from the nucleus and cytoplasm by using the protein extraction kit (Beyotime Biotechnology, Cat# P0027). Proteins were separated by SDS-PAGE electrophoresis using standard methods, transferred to PVDF membrane, and immunoblotted with specific antibodies overnight at 4 °C against PPARγ (detect both PPARγ1 and PPARγ2, Engene, Cat# E2A6073, 58 kDa, RRID:AB_2861246 ), MYOCD (Abcam, Cat# Ab107301,100 kDa, RRID:AB_11128102 ), OPN (Proteintech, Cat# 22952-1-AP, 44 kDa, RRID:AB_2783651 ), MMP2 (Proteintech, Cat# 10373-2-AP, 64 kDa, RRID:AB_2250823 ), Col I (Engene, Cat# E110154C, 139 kDa, RRID:AB_2861247 ), Col III (Abcam, Cat# Ab7778, 139 kDa, RRID:AB_306066 ), p65NF-κB (Engene, Cat# E011014, 65 kDa, RRID:AB_2861239 ), phosphorylated p65NF-κB (Engene, Cat# E1A2006V, 65 kDa, RRID:AB_2861248 ), VCAM1 (Engene, Cat# E90279, 72 kDa, RRID:AB_2861237 ), ICAM1 (Engene, Cat# E301187, 82 kDa, RRID:AB_2861238 ), NFAT4 (Abcam, Cat# Ab93628, 117 kDa, RRID:AB_10714571 ), GAPDH (Engene, Cat# E1C604, 37 kDa, RRID: AB_2814765 ), Histone H3 (Engene, Cat# E11-0434B, 17 kDa, AB_2861240 ), followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody (Sino Biological Inc., Cat# SSA003, RRID:AB_2814815 ) 1 hr at room temperature. Proteins were visualized with an X-ray film system (Fujifilm, Japan) or ChemiDoc™ Touch System (Bio-Rad, USA). Band density was quantified by NIH ImageJ software (RRID:SCR_003070 , https://imagej.net/) and normalized to GAPDH or Histone H3.