Anti p smad3

Western blotting was carried out as previously described [27 (link)]. The primary antibodies used were anti-N-Cadherin (BD Biosciences, Breda, Netherlands #610920), anti-α-Smooth Muscle Actin (Sigma, Zwijndrecht, Netherlands #A2547), anti-Snail (Cell Signaling, Leiden, Netherlands #3879), anti-Smad2 (BD Biosciences, Breda, Netherlands #610842), anti-p-Smad2 (Cell Signaling, Leiden, Netherlands #3108), anti-Smad4 (Santa Cruz #sc7966), anti-TGFβRI (Santa Cruz, Heidelberg, Germany #sc 398), anti-TGFβRII (Santa Cruz, Heidelberg, Germany #sc-400), anti-Smad3 (Epitomics, Duiven, Netherlands #1735–1), anti-p-Smad3 (a kind gift from Dr Edward B Leof, Mayo Clinic, Rochester, Minnesota) and anti-β-actin (Sigma, Zwijndrecht, Netherlands #A5441). All the secondary antibodies were from Sigma, Zwijndrecht, Netherlands. Western quantification was performed using image J software.

5 × 10⁵ lens epithelial cells were grown in a 6-well plate in the presence of serum-containing media, followed by serum starvation for 6–8 h. Post starvation, cells were treated with inhibitors for p38 and ERK pathways (10 μM) or with scFv antibodies and TGF-β2 (2 ng/ml) was added 1 h after the addition of the reagents, and was further incubated for 2 h. At the end of the incubation, cells were washed twice with PBS, harvested and lysed in 100 μl of ice cold RIPA buffer (25 mM Tris-HCl [pH 7.6], 1 mM Na₃VO₄, 1% sodium deoxycholate, 150 mM NaCl, 0.1% SDS, 1% NP-40) with a cocktail of protease inhibitors. Cell lysate was separated on 10% SDS-PAGE, transferred on the nitrocellulose membrane, blocked with 2% BSA for 1 h and probed with primary anti-pFAK (Y397, 1:500; Sigma Chemicals), anti-pSMAD3 (Y208; Sigma Chemicals), anti-pERK (Th202/Tyr204; Cell Signaling), or pp38 (Th180/Tyr182; Cell Signaling) antibodies, followed by anti-rabbit-HRP conjugate antibody (1:10,000). The membrane was developed using ECL reagent (Bio-Rad) and acquired on FluorChem E Imaging System. For evaluation of the corresponding total protein levels of each molecule, stripping and re-probing of the membrane was carried out with anti-FAK, Anti-SMAD3, anti-ERK and anti-p38 antibodies. Equal loading was ensured by probing with anti-β-actin antibody.

RIPA buffer (MilliporeSigma; Merck KGaA) was used to extract total proteins from cultured 22Rv1 or PC-3 cells. Proteins were quantified with a BCA method and separated by 12% SDS-PAGE, and transferred onto PVDF membranes (GEHealthcare). The membranes were blocked in 5% non-fat dry milk diluted with TBST (Tris–HCl 20 mmol/L, NaCl 150 mmol/L, pH 7.5, 0.1% Tween 20) at room temperature for 1 h and washed with TBST for three times. Subsequently, specific primary antibodies (anti-Vimentin, anti-N-cadherin, anti-a-SMA, anti-E-cadherin, anti-Claudin 1, anti-GAPDH, anti-p-Akt, anti-Akt, anti-p-mTOR, anti-mTOR, anti-PTEN, anti-p-Smad2, anti-Smad2, anti-p-Smad3, anti-Smad3, anti-p-Smad2/3, anti-Smad2/3, and anti-laminB, Sigma, USA) were incubated with the membranes at 4 °C overnight. Next, the membranes were washed with TBST for three times and incubated with the secondary antibodies at room temperature for 2 h. Proteins were visualized using chemiluminescence using ECL luminescence reagent (Absin Bioscience Inc., Shanghai, China). The band intensities were quantified using Image-Pro Plus 6.0 (Media Cybernetics, Inc.). GAPDH was used as a loading control for normalization. The primary antibodies and secondary antibodies used in western blotting are included in Table 1.