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.