Phospho smad1 smad5 smad8 antibody

Representative three 1-mm-diameter cores from each tumor taken from the formalin-fixed paraffin embedded tissues were selected by morphology typical of the diagnosis. Immunohistochemical (IHC) staining was performed on serial 5-micrometer-thick tissue sections cut from the tissue microarray (TMA). IHC staining of FSTL1, BMP4, and Smad4 was performed using an automated immunostainer (Ventana Discovery XT autostainer, Ventana, USA). The antigens were retrieved by heat-induced antigen retrieval for 30 minutes with TRIS-EDTA buffer. The slides were stained with a polyclonal rabbit FSTL1 antibody (1:250; GeneTex, Taiwan), a polyclonal rabbit BMP4 antibody (1:300; GeneTex, Taiwan), and a polyclonal rabbit Smad4 antibody (1:500; Proteintech, USA). For phospho-Smad1/Smad5/Smad8 (p-Smad1/5/8), manual IHC staining was performed. Briefly, the slides were submitted to heat-induced antigen retrieval for 10 minutes with DAKO antigen retrieval buffer (pH 6) and incubated at 4 °C overnight with a polyclonal rabbit phospho-Smad1/Smad5/Smad8 antibody (1:50; Cell Signaling, USA) and then visualised using the 3, 3′-diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories, USA).

The three hiPSC lines were incubated with or without BMP4 (50 ng/mL) for 4 hours. Protein was extracted as previously described [16 (link)] and concentration was determined by BCA assay (Pierce). Fifteen μg of protein was separated by SDS- PAGE (Invitrogen) and transferred to Hybond-P PVDF membrane (Amersham). The membrane was blocked with 5% non-fat dried milk in TBST for 30 minutes, incubated with diluted PhosphoSmad1/Smad5/Smad8 antibody (9511S, Cell Signaling Technology) in 5%(w/v) BSA, 1xTBST at 4°C with gentle shaking overnight, then incubated with HRP-conjugated donkey anti-rabbit IgG (1:2,000 in TBST, Cell Signaling Technology) at ambient temperature for 1 hour. Reactive protein bands were visualized using ECL plus Western blotting detection reagents (GE Healthcare) and band intensities were calculated using the Bio Rad Chemi Doc XRS imaging system. Beta-actin (13E5) Rabbit mAB (4970, Cell Signaling Technology) was used for a loading control.

Cell extracts were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane following the manufacturer’s protocol (Bio-Rad). Membranes were incubated with 5% nonfat milk or 3% BSA (Sigma) in TBST for 60 min. Primary and secondary antibodies used for the western-blot assays are as follows: Phospho-Smad1/Smad5/Smad8 antibody (Cell Signalling, Ref. 9511 and Ref. 13820), Smad1/5/8 (N-18)-R antibody (Santa Cruz Biotechnology, Ref. sc-6031-r), Neuronal Class III β-Tubulin antibody (Covance, Ref. mms-435p), Glial Fibrillary Acidic Protein antibody (Dako, Ref. z0334), Goat Doublecortin (C-18) antibody (Santa Cruz Biotechnology, Ref. sc8066), Phospho-LRP6 (Ser1490) antibody (Cell Signalling, Ref. 2568), β-actin antibody (Sigma-Aldrich, Ref. a5441), Donkey anti-Rabbit ECL 1:50000 (Amersham, Ref. na934), Sheep anti-Mouse ECL 1:50000 (Amersham, Ref. na931), Donkey anti-Goat HRP 1:10000 (Santa Cruz Biotechnology, Ref. sc-2020).