P smad2 3

Cellular proteins were isolated from hepatic tissues in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% Nonidet P-40, 1 mM EDTA) (Millipore, Billerica, MA, USA) containing a protease inhibitor cocktail (Roche) and quantified with bicinchoninic acid protein concentration assay kit (Beyotime Biotech, Beijing, China). Protein of each sample was separated by electrophoresis in 10% SDS-PAGE with a Bio-Rad electrophoresis system (Hercules, CA, USA). After electrophoresis, the resolved protein was transferred onto PVDF membranes (Millipore). Membranes were then incubated with primary rabbit antibodies: α-SMA (1:1000, UCallM biotech Co., Ltd, Wuxi, China), MyD88 (1:1000, UCallM biotech Co., Ltd, Wuxi, China), p-NF-κB (1:1000, UCallM biotech Co., Ltd, Wuxi, China), TGF-β₁ (1:1000, Affinity Biosciences, OH, USA), p-Smad2/3 (1:1000, Proteintech, Wuhan, China), BAMBI (1:1000, Proteintech, Wuhan, China) and β-actin (1:2000, Santa Cruz Biotechnology, CA, USA) at 4 °C overnight. The corresponding horseradish-peroxidase-conjugated secondary antibodies (anti-rabbit IgG, Santa Cruz Biotechnology, CA, USA. 1:2000 dilutions) were incubated for 2 h at room temperature. The protein bands were visualized using enhanced chemiluminescence reagents (Millipore). The optical density of each protein in each sample was normalized against β -actin.

As our laboratory described previously [6 (link)], the culture medium was discarded and cells were washed with phosphate buffer solution (PBS) three times. RIPA lysis buffer (Beyotime, China) was used to extract protein, and the BCA protein assay kit (Beyotime, China) was used to quantify protein concentration. The protein was separated with 10%–15% SDS-PAGE gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Then, the membranes were immersed in PBST (phosphate buffered solution containing 0.1% Tween-20) with 5% skim milk powder for 1 h at room temperature to block nonspecific antigen. After being washed with PBST, the membranes were incubated with primary antibodies against PFKFB3 (1:1000, Proteintech, USA), vimentin (1:1000, Proteintech, USA), E-cadherin (1:1000, Proteintech, USA), N-cadherin (1:1000, Proteintech, USA), p-smad2/3 (1:1000, Proteintech, USA), smad2/3 (1:1000, Proteintech, USA), TGF-β (1:1000, Proteintech, USA), β-actin (1:1000, Proteintech, USA) for the whole night at 4°C. The membranes were washed with PBST three times the next day and incubated with the secondary antibodies (1:10,000, Proteintech, USA) for 1 h at room temperature. After being washed with PBST three times, the protein expression level in membranes can be detected with an enhanced chemiluminescence detection system (Amersham Imager 600, USA).