Anti p erk1 2

Total proteins, membrane proteins and cytoplasmic proteins were extracted (Beyotime,
Haimen, China) and equal amounts of proteins were run on SDS-PAGE. After electrophoresis,
the proteins were transferred onto Millipore PVDF membranes (Billerica, Massachusetts,
USA). The membranes were immersed in 5% nonfat milk and incubated with anti-Angiotensin
converting enzyme (ACE) (1:200) (Santa Cruz, Dallas, Texas, USA), anti-Angiotensin II type
1 receptor (AT1 receptor) (1:1,000) (Abcam, Cambridge, MA, USA), anti-periostin (1:200)
(Santa Cruz), anti-collagen I (1:400) (Wuhan Boster, Wuhan, China), anti-TGF-β1 (1:200)
(Santa Cruz, USA), anti-Smad2/3 (1:200) (Santa Cruz), anti-p-ERK1/2 (1:500) (Bioss,
Beijing, China), anti-ERK1/2 (1:500) (Bioss), anti-GLUT4 (1:200) (Santa Cruz), anti-p-Akt
(1:200) (Santa Cruz) and anti-Akt (1:200) (Santa Cruz, USA) primary antibodies overnight.
After washing with TBS-T buffer, the membranes were incubated for 45 min with horseradish
peroxidase (HRP)-conjugated secondary antibody (1:5,000) (Beyotime) diluted in 5% nonfat
milk. The proteins were developed using ECL reagent in the dark and quantified using
Gel-Pro Analyzer (Media Cybernetics, Inc., Rockville, MD, USA).