P smad2 3

Automated immunoblot was carried out with Simple Wes (Protein Simple, USA) as described by the manufacturer. In brief, 1.5 μg total protein (cell lysate or exosome specimens) was loaded into a Wes assay plate, alongside antibody diluent (Protein Simple); primary antibodies targeting DSP (Santa Cruz, USA), DMP1, CD9, CD63, p-NFκΒ, IκB-a, Myd88, TLR4, IKBKB, BMP2, BMPR1, TGFβ1, TGFβ3, TGFR1, p-Smad2/3, p-Smad1/5/9, Smad4, β-actin, and β-Tublin (Affinity, USA); and anti-rabbit/mouse secondary antibodies (Protein Simple) and streptavidin-HRP. The compass software (Protein Simple) was employed for data analysis.

The tissue around the material was frozen and milled in liquid nitrogen, and the cell protein was extracted with RIPA lysis buffer. After electrophoretic separation, the proteins were transferred to PVDF membranes, and the membranes were blocked with 5% skimmed milk powder solution at room temperature for 1 h. Next, the membranes were incubated with primary antibody at 4°C overnight. The membranes were washed with TBST 3 times for 10, the secondary antibody (antibody dilution ratio of 1:8,000) was added, and the membranes were incubated for 1 h. The membranes were then washed with TBST 3 times for 10 min. Luminescent reaction solution was added, and the protein expression content was determined. The primary antibodies were as follows: Smad2/3 (1:500, Affinity, United States), vimentin (1:500, Proteintech, China), α-SMA (1:500, Proteintech, China), Col-1A1 (1:500, Novus, United States), TGF-β1 (1:500, Affinity, United States), TβRI (1:500, Abcam, United States), TβRII (1:500, Abcam, United States), Smad2/3 (1:500, Affinity, United States), p-Smad2/3 (1:500, Affinity, United States), and β-actin (1:1,000, Abcam, United States).

Affinity Biosciences: p-SMAD1/5/8 at Ser463 + Ser465 (AF8313; 1:1000), SMAD1/5/8 (AF0614; 1:1000); p-SMAD2/3 at Thr8 (AF3367; 1:1000); SMAD2/3 (AF6367; 1:1000); C/EBPα (AF6333; 1:1000); p-ERK1/2 at Thr202/Tyr204 (AF1015; 1:1000); ERK1/2 (AF0155; 1:1000); p-p65 at Ser536 (AF2006; 1:1000); p65 (AF5006; 1:1000); p-IKKα/β at Ser180/Ser181 (AF3013; 1:1000); IKKα/β (AF6014; 1:1000). Sangon Biotech: p-ACC at Ser79 (D155180; 1:1000); ACC (D155300; 1:1000). Abcam: BMP8B (ab181253; 1:1000). CWBIO: Goat Anti-Rabbit IgG, HRP Conjugated (CW0103S; 1:4000). Bioss: PPARγ (bs-4590R; 1:1000); p-p38 MAPK at Thr180 + Tyr182 (bs-2210R; 1:1000); p38 MAPK (bs-0637R; 1:1000); β-actin (bs-0061R; 1:2000); p-JNK at Thr183 + Tyr185 (bs-1640R; 1:1000); JNK (bs-2592R; 1:1000).