Tgfbr2

Paraffin embedded slides were deparaffinized and rehydrated followed by antigen-retrieval using citric acid buffer. Endogenous peroxidase was deactivated by H₂O₂. Slides were blocked using 10% goat serum and incubated with the corresponding primary antibodies overnight at 4 °C. After incubation with secondary antibodies for 45 min at room temperature, DAB staining (Thermo Fisher Scientific) was used to detect the antigen–antibody binding. The primary antibodies used were: GABPA (ProteinTech) and TGFBR2 (Abcam). The slides were examined by two of the co-authors (ZF and NZ) and mean values of GABPA and TGFBR2 positive cells were presented based on the results from two observers. For each slide, a total of 200 cells in two fields were analyzed.

Proteins were extracted using Pierce RIPA Buffer (Thermo Scientific) with 1% Phenylmethanesulfonyl fluoride (Sigma-Aldrich) and quantified with DC Protein Assay (Bio-Rad). Thirty µg of proteins were separated in Mini-PROTEAN TGX Gels (Bio-Rad) and transferred to PVDF membranes using Trans-Blot Turbo Transfer Pack (Bio-Rad). Membranes were blocked with 5% non-fat milk diluted in TBST, and then incubated with primary antibodies and secondary antibodies before imaged with Clarity Max Western ECL Substrate (Bio-Rad, 1,705,062) and ChemiDoc MP Imaging System (Bio-Rad). The following primary antibodies were used: GABPA (Santa Cruz), L2HGDH (Novus), LDHB (Santa Cruz), MDH2 (Santa Cruz), cMYC (Santa Cruz), TGFBR2 (Abcam), SMAD2/3 and p-SMAD2/3 (Sigma-Aldrich), CDKN1A (Cell Signaling Technologies), CDH1 (Cell Signaling Technologies), vimentin (Cell Signaling Technologies) and Actin (Santa Cruz). Secondary antibodies include Goat Anti-Mouse IgG (H + L)-HRP Conjugate and Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (Bio-Rad).

Activin A was reconstituted in PBS; TGFβ1 in 4 mM HCl according to the manufacturer’s instruction (both R&D, Minneapolis, MN, USA). Final concentrations used were 25 ng/ml and 10 ng/ml, respectively, as previously described [31 (link), 49 (link)–51 (link)]. For inhibition of PI3K, we used LY294002 and for inhibition of MEK1/2, U0126 (both Cell Signaling Technology, Danvers, MA, USA). For immunoprecipitation and Western blotting, we used antibodies against ACVR1B (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ACVR2A (customized by Yenzym, San Francisco, CA, USA), p85 (# 4292), TGFBR1 (# 3712) or TGFBR2 (# 3713, all Cell Signaling), p21 (# sc-65595, Santa Cruz), pan-Akt (# 8805, Abcam, Cambridge, MA, USA), GAPDH (# sc-47724, Santa Cruz), E-Cadherin (# 3195), vimentin (# 3390), pAkt Ser473 (# 4060) and pAkt Thr308 (# 13842, all Cell Signaling). For immunohistochemical analyses, we used p21 (# sc-817, Santa Cruz) ACVR2A (# ab10595), TGFBR2 (# ab78419), pAkt Ser473 (# ab81283, abcam), and pERK1/2 (# ab50011, all Abcam).

HIF-1α, HIF-2α content was detected by western blot. Cell homogenate protein was prepared using loading buffer, RIPA lysis buffer as well as protease inhibitor (Beyotime Institute of Biotechnology, Jiangsu, China). Cell homogenate was then boiled for 5 min. Samples were centrifuged at 17,000 rpm, 4°C. The concentrations of proteins were measured using the BCA protein assay kit (Beyotime Institute of Biotechnology).
Protein samples of interest went electrophoresis and then were transferred to polyvinylidene fluoride membrane. Membrane was blocked in TBST (Tween Tris-buffered saline) buffer containing 5% low fat milk powder, then incubated with primary antibodies respectively at 4°C overnight. Primary antibodies included HIF-1α (diluted at 1:1000, Abcam, Cambridge, UK), HIF-2α (1:500, Abcam), Bcl-2 (1:2000, Proteintech Group, Chicago, USA), Bax (1:3000, Proteintech Group), CD44 (1:1000, Proteintech Group), TGFBR2 (1:500, Abcam), GAPDH (1:5000, Cell Signaling, Beverly, MA, USA). Then, membranes were incubated with corresponding HRP-polymerization secondary antibodies diluted at 1:5000 for 2 h at room temperature. Protein bands were visualized by ECL detection system, and ChemImager5500V2.03 scanning software. The integrated light density values (IDV) were calculated with software FluorChem2.0.

Cell lysates were prepared with RIPA buffer (Cell Signaling, #9806), supplemented with 1 mM PMSF, 1 mM NaF, protease inhibitor cocktail (Roche, #04 693 132 001), and phosphatase inhibitor cocktail (Roche, #04 906 845 001). SDS–PAGE gels were transferred onto nitrocellulose membranes.
We used the following primary antibodies against: Phospho-SMAD2 (Ser465/467) (Cell Signaling, #3108), SMAD2 (Cell Signaling, #3103), β-Actin (Cell Signaling, #3700), TGFBR1 (Santa Cruz, #sc-398), TGFBR2 (abcam, #ab184948), and integrin β1 (Cell Signaling, #9699). All Western blot membranes were incubated with primary antibodies overnight at 4 °C. Secondary antibodies used for Western blotting were anti-rabbit IgG (H+L) DyLight 800 (Cell Signaling, #5151) and anti-mouse IgG (H+L) DyLight 680 (Cell Signaling, #5470). Western blot images were acquired and quantified using Odyssey CLx Imaging System (LI-COR Biosciences, #9140).