Tβrii

Proteins were isolated and protein concentration was measured using BCA protein assay. The proteins were subjected to SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride membrane (Millipore Corp, Massachusetts, USA). After blocking, membranes were incubated with primary antibodies (β-actin, Type I Collagen, Type III Collagen, Smad2/3 and TβRII, Santa Cruz Biotechnology, Dallas, Texas, USA; p-Smad2/3, Cell Signaling Technology, Danvers, USA) overnight at 4 °C followed by incubation with AP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, USA). The proteins were visualised with BCIP/NBT kit (Invitrogen, Carlsbad, CA, USA).

These antibodies were used in the present investigation: Kindlin-1 (Millipore, MA), c-myc (Snail (Millipore), E-cadherin (Abcam), N-cadherin (Epitomics), p-Smad3 (Abcam), Smad2 (Epitomics), Smad3 (Epitomics), SARA (Epitomics), TβRI (Santa Cruz, CA), TβRII (Santa Cruz, CA), Vimentin (Epitomics), YY-1 (Santa Cruz, CA), Actin (Santa Cruz), or Flag (Sigma-Aldrich, St. Louis, MO).

Antibodies against Smad2/3 and pSmad2/3 antibodies were obtained from Cell Signaling Technology (CST, USA). Antibodies against TEAD1, TβRII, GFP, flag, Ub, GAPDH, c-Cbl siRNA, and TEAD1 siRNA were ordered from Santa Cruz Biotechnology. The secondary fluorescent antibodies and H&E staining kit were ordered from Abcam. Calcein labeling, osmium tetroxide, and lipofectamine^™ 3000 were purchased from Fisher Scientific^™. Dexamethasone, L-ascorbic acid, and β-glycerophosphate were ordered from Sigma. Plasmids pcDNA3.1-flag and pcDNA3.1-flag-TβRII, GFP-IFT20, HA-Ub were obtained from Addgene.

Formalin-fixed paraffin-embedded microarrays of breast cancer tissues were obtained from US Biomax (BC081120). Primary antibodies specific to p-SMAD2 (1:50; Cell Signalling 3108), FAF1 (1:200; Bethyl), TβRII (1:100; Santa Cruz) and p-Akt Ser 473 (1:200; Cell Signalling #9271) were used for immunohistochemical staining. The quantification of staining was expressed as an H score. The H score was determined by the formula 3 × the percentage of strongly staining cells+2 × the percentage of moderately staining cells+the percentage of weakly staining cells, yielding a range of 0 to 300.

Total proteins were isolated from the whole frozen aorta using lysis buffer as previously described [28 (link)]. Afterwards, proteins were quantified using bicinchoninic acid assay (BCA) method (Thermo Fisher Scientific). A total 50 ug of proteins were loaded and separated on 10% polyacrylamide-SDS gels under reducing conditions. At the end of electrophoresis, proteins were transferred to nitrocellulose membranes (Amersham Bioscience, Buckinghamshire, UK). Membranes were blocked in TBS containing 0.1% Tween20 (TBST) and 5% dry non-fat milk (1 h at room temperature) and incubated with the different primary antibodies overnight at 4 °C. Next day, membranes were washed 10 min three times with TBST and incubated 1 h with the appropriate HRP (horseradish peroxidase)-conjugated secondary antibody (anti-rabbit, GENA934, anti-mouse, GENA931, Sigma Chemical [1/2000]) at room temperature. ECL kit (Amersham Bioscience) was used to develop. Results were analyzed by LAS 4000 (GE Healthcare Systems, Chicago, IL, USA) and the quantification of the bands density was done by using the Quantity One software (Bio-Rad, CA, USA). The following primary antibodies were employed p-SMAD2 (#3108, Cell signaling (1/1000)), p-SMAD3 (ab52903; abcam; (1/1000)), TβRII ((sc17792; Santa Cruz Biotechnologies; 1/300)) and TβRI (sc518086; Santa Cruz Biotechnologies; [1/300]).