Anti p smad2

RIPA buffer was used to extract total protein from HUFs, human and rat urethral tissues. BCA protein assay (Thermo Fisher Scientific) was performed to detect the protein concentration. Equal amounts of protein (20 µg) were separated by 10% SDS-PAGE gels and transferred to PVDF membrane (Millipore, USA). Next, the membranes were blocked in 5% fat-free milk for 2 h, and incubated with the following antibodies at 4 °C overnight: anti-Wnt3a (1:1000, Abcam), anti-β-catenin (1:1000, Abcam), anti-DKK1(1:1000, Abcam), anti-collagen I (1:1000, Abcam), anti-collagen III (1:1000, Abcam), anti-α-SMA (1:1000, Abcam), anti-p-GSK-3β (1:1000, Abcam), anti-GSK-3β(1:1000, Abcam), anti-p-Smad2(1:1000, Abcam), anti-p-Smad3 (1:1000, Abcam), anti-Smad2/3 (1:1000, Abcam) and anti-GAPDH (1:5000, Abcam, Cambridge, UK). Membranes were cut horizontally based on the differences of molecular weight. Then membranes washed with TBST (Beyotime Biotechnology, Shanghai, China) and incubated with corresponding secondary antibodies (goat anti-rabbit IgG H&L (HRP), 1:10000; rabbit anti mouse IgG H&L (HRP), 1:10000; Abcam, USA) at room temperature for 1 h. Additionally, membranes were stripped with Western Blot Stripping Buffer (Cell Signaling Technology), and re-probed with target proteins. Target blots signals were observed by an enhanced chemiluminescence-detecting kit (Thermo Fisher, MA, USA).

Expression of SMAD7, phosphorylated (p)-SMAD2, RORγT, and TGF-β were detected in both the human lung tissue homogenates samples and in the sorted CD4⁺ mouse lymphocytes using standard Western blot techniques. Isolated proteins were transferred onto a nitrocellulose membrane, and immunoblotting was performed using mouse monoclonal anti-SMAD-7 (R&D Systems, USA, dilution 1:1000), anti-p-SMAD2 (Abcam, USA, dilution 1:1000), anti-p-SMAD3 (Abcam, USA, dilution 1:1000), anti-TGF-β (Abcam, USA, dilution 1:1000), anti-ROR-γT (Abcam, USA, dilution 1:1000), anti-β-actin (Proteintech, USA, dilution 1:5000), and conjugated HRP-labeled secondary mouse or rabbit antibodies (Proteintech, USA). Images were captured using the Quantity One Analysis Software (Bio-Rad Laboratories Inc, Hercules, CA, USA).

Uterine tissue and ESCs protein were extracted using RIPA lysate (Beyotime, Shanghai, China). After 15 min of lysis on ice, 12 000 rpm/10 min, the supernatant was taken to quantify the protein (BCA protein detection kit 23225, Thermo Fisher Scientific, USA). A 30 μg protein sample was added to the loading buffer and denatured at 99°C for 10 min, separated on a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (Amersham, St Albans, UK). Anti-TGF-βI (ab215715, Abcam, Cambridge, UK; 1:500), anti-Smad2/3 (Abcam, Cambridge, UK; 1:200), anti-α-SMA (Abcam, Cambridge, UK; 1:500), anti-pSmad2 (Abcam, Cambridge, UK; 1:200) and anti-pSmad3 (Abcam, Cambridge, UK; 1:200) in Incubate overnight at 4°C. Then, incubate the membrane with anti-rabbit secondary antibody (ZSJQ-Bio, Beijing, China; 1:2000) at room temperature for 1 h. Protein band was visualized using a chemiluminescence kit (New Cell Molecular Biotechnology Co., Ltd, Suzhou, China). ImageJ is used to quantify the intensity of the bands.

EVs and cells were lysed with RIPA lysis buffer including protease and phosphatase inhibitors (Boyotime, Beijing, China). Protein concentrations were determined using the Pierce™ BCA Protein Assay kit (Thermo Scientific, USA). Western blots were performed as previously described [46 (link)]. The primary antibodies used in the experiments included anti-CD63 (1:500, Santa Cruz, CA, USA), anti-TSG101 (1:500, Santa Cruz, CA, USA), anti-GM-130 (1:500, Santa Cruz, CA, USA), anti-E-cadherin (1:1000, ABclonal, Wuhan, Hubei, China), anti-N-cadherin (1:1000, Affinity Biosciences, Cincinnati, OH, USA), anti-Snail (1:1000, ABclonal, Wuhan, Hubei, China), anti-α-SMA (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-PTEN (1:1000, Cell Signaling Technology, USA); anti-PI3K (1:1000, Abcam, USA), anti-Akt, anti-p-Akt (Ser473, 1:1000, Abcam, USA), anti-p-STAT6 (Y641, 1:1000, Abcam, USA), anti-p-Smad2 (1:1000, Abcam, USA), anti-GAPDH (1:1000, ABclonal, Wuhan, Hubei, China), and anti-β-actin (1:1000, Abcam, USA). ImageJ version 1.48 was used to analyze Western blot results [47 (link)].

1× Nupage LDS lysis buffer supplied by Life Technologies, Carlsbad, CA, USA was used for the lysis of regulatory CD4⁺CD25⁺ T cells. The addition of phosphatase inhibitors supplied by Life Technologies was left as a choice. After lysis, cellular incubation was carried for 10 min at 95°C. The protein concentrations were quantified using the BCA (Life Technologies). A 10%–15% SDS‐PAGE was performed to extract equal protein quantities extracted from the lysed cells (Boster), followed by blotting onto nitrocellulose membranes. 3% BSA solution (Boster) was used to block the membranes for 2 h at 25°C, followed by incubation with primary antibodies (1:1000) at 4°C until the next morning. Rabbit antibodies including anti‐mouse anti‐Smad2, anti‐p‐Smad2, anti‐Smad3, and anti‐p‐Smad3 were supplied by ABCAm. Afterward, horseradish peroxidase‐conjugated with goat anti‐rabbit IgG (ABCAm) secondary antibodies were poured onto the membranes followed by an incubation of 1.5 h at room temperature. Lab Works^tm imaging and analysis system by UVP was used for data collection and processing.