For each sample, 20 μg of protein was separated by SDS/PAGE and transferred to PVDF membranes.30 The membranes were blocked for 1 hour at room temperature with 5% powdered skim milk in Tris‐buffered saline pH 7.4 with 0.05% Tween (TBST) and incubated with antibodies for TGFBR2 (#SC400; 1:1000; Santa Cruz Biotechnology, Dallas, TX), phospho‐SMAD2 (S465‐467, #3101S; 1:1000; Cell Signaling, Danvers, MA), SMAD2 (#3122S; 1:1000; Cell Signaling), phospho‐ERK1/2 (T202‐Y204, #9101S; 1:1000; Cell Signaling), ERK1/2 (#9102S; 1:1000; Cell Signaling), phospho‐p38 (T180‐Y182, #4511S; 1:2000; Cell Signaling), p38 (#9212S; 1:2000; Cell Signaling), or β‐actin (#A5316; 1:5000; Sigma, St. Louis, MO). Antibodies were diluted in TBST with 5% milk and incubated overnight at 4°C. Blots were then washed 3 times (5 minutes each) in TBST and incubated with HRP‐conjugated secondary antibody (goat antirabbit or antimouse; Biorad, Hercules, CA; #170‐6515 or #170‐6516; 1:3000‐1:5000 in TBST with 5% milk) for 1 hour at room temperature. Bound antibodies were detected using enhanced chemiluminescence (ECL) (Thermo, Rockford, IL). Band density was analyzed by densitometry, using the Image J software Version 1.48 (NIH, Bethesda, MD).
Tgfbr2
Manufactured by Santa Cruz Biotechnology
TGFBR2 is a gene that encodes the transforming growth factor beta receptor type-2 protein. This protein is a transmembrane serine/threonine protein kinase that forms a heteromeric complex with the transforming growth factor beta type-1 receptor upon ligand binding. This complex then phosphorylates and activates SMAD transcription factors, which regulate the transcription of target genes.
Automatically generated - may contain errors
Lab products found in correlation
Tgfbr1, by Santa Cruz Biotechnology (2 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Phospho p38t180 y182, by Cell Signaling Technology (1 mentions)
Phospho erk1 2t202 y204, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Smad2, by Cell Signaling Technology (1 mentions)
Phospho smad2, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence solution, by Cytiva (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
Gapdh, by OriGene (1 mentions)
Smad3, by Santa Cruz Biotechnology (1 mentions)
Phosphor smad3, by Cell Signaling Technology (1 mentions)
Smad4, by Santa Cruz Biotechnology (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
8 protocols using tgfbr2
1
Western Blot Analysis of Signaling Pathways
2
Western Blot Analysis of TGF-β Signaling
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Cells were lysed directly in 4 × SDS sample buffer at 60–80% confluence. Lysates were subjected to standard SDS–PAGE. Bands were detected using enhanced chemiluminescence solution (Amersham). Secondary antibodies used throughout were horseradish peroxidase-conjugated polyclonal goat anti-mouse Ig (Dako, P0448, 1:2,000) and horseradish peroxidase-conjugated polyclonal goat anti-rabbit Ig (Dako, P0260, 1:2,000). Primary antibodies were PO4-SMAD3 (Abcam, 52903, 1:1,000), SMAD3 (Cell Signaling, 9523, 1:1,000), TGFBR1 (Santa Cruz, 398 (V22), 1:500) and TGFBR2 (Santa Cruz, 17792, (E6), 1:500). For TGFBR2 western blottings, lysates were prepared directly from transfected cells using the Dual-luciferase cell lysis buffer (Promega, UK). For TGFBR1 western blottings, parallel transfections to the luciferase assays were performed and samples were lysed directly in 4 × SDS sample buffer. Original uncropped western blot scans are also provided (Supplementary Fig. 15 ).
3
Probing Cell Signaling Pathways with Western Blot
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Antibodies used for western blotting include TGFBR2 (Santa Cruz #sc-400, 1:1000), SMAD3 (Santa Cruz #sc-8332, 1:1000), SMAD4 (Santa Cruz #sc-1909-R, 1:1000), phosphor-SMAD3 (Cell Signaling Technology #9520, 1:2000), and GAPDH (ORIGENE #TA802519, 1:5000). The method of western blot was described in detail in ref. 22 (link).
4
Western Blot Analysis of EMT Markers
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For western blot analysis cells were lysed using RIPA buffer containing PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Protein concentration was determined by Bradford assay (Bio‐Rad). Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences). The following antibodies were used: E2F1 (KH‐95; 1:500), EGFR (1:250), FGFR1 (Flg C15; 1:1000), TGFBR1 (V22; 1:1000), TGFBR2 (L21; 1:1000), Vimentin (V9; 1:1500), ZEB1 (H‐102; 1:1000), Snail (H-130; 1:500), SMAD2/3 (FL-425; 1:1000), and c-SRC (1:500) from Santa Cruz, E‐Cadherin (1:1500), and NFKB1 (C22B4; 1:1500) from Cell Signaling; N-Cadherin (610921; 1:1500) and FN1 (1:1000) from BD Bioscience, Actin (Sigma; 1:4000) and their corresponding HRP‐conjugated secondary antibodies (Pierce; 1:2000). Detection of HRP activity was performed with the ChemiDoc TouchTM Imaging System (BioRad) using ECL Plus (Amersham) or Super Signal West Femto (Thermo Scientific) Western Blotting Detection Reagents (GE Healthcare). Uncropped pictures of the immunoblots are shown in Supplementary Figs 9 , 10 , and 11 .
5
Colon Cancer Tissue Immunostaining
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Human colon cancer tissue microarray slides were obtained from AccuMax ISU ABXIS and contained 32 colon cancer specimens. After baking and deparaffinization, the slides were boiled in a pressure cooker filled with 10 mmol/L sodium citrate (pH 6.0) and then immunostained with antibodies targeting phospho‐STAT3 (Ser‐727; 1:25; Cell Signaling Technology) and TGFBR2 (1:50; Santa Cruz Biotechnology). Spots were evaluated by estimating the intensity of tumor cells. Samples were considered positive if 30% or more of the tumor cells were immunostained.
6
Morphological Analysis of Mouse Mandibular Molars
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Mouse mandibles (n = 4 mice/genotype at P20, P30, and P120) were sectioned and were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) overnight, then were decalcified and embedded in paraffin, and sectioned at 6 μm. Histological structures of mouse mandibular first molars were visualized using hematoxylin-eosin staining protocol. For immunohistochemical staining, antigen retrieval was performed in citrate buffer (pH 6.0) using a pressure cooker (Biocare Medical, Concord/California, biocare.net , DC2008). The primary antibodies used in the immunohistochemistry analyses were Tgfbr2 (Santa Cruz, sc-17792), β-catenin (BD Transduction Laboratories, 610153). Bound antibodies were visualized with diaminobenzidine, and sections were counterstained with hematoxylin, and observed under microscopy (Nikon, Eclipse E600).
7
Immunohistochemical Analysis of Smooth Muscle Cells
8
Western Blot Protein Expression Analysis
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Cells/tissue were harvested, washed in PBS, and resuspended in RIPA buffer (CST-9806) containing proteinase inhibitors (Roche) and quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher). Protein lysates diluted in 4 X Laemmli Sample Buffer (Bio-Rad 161–0747) were loaded onto Bio-Rad 4–20% precast gels. Following electrophoresis, proteins were transferred to a pre-activated PVDF membrane using the Trans-Blot®Turbo™ Transfer System and visualized using ECL (Bio-Rad Chemidoc). Antibodies used in this study are DUSP4 (CST#5149), Cyclin D1 (CST#2978), Cyclin E1 CST#4129), ATF7IP (Sigma#16,578), ATF7IP (Sigma#HPA023505), ADCY3 (Abcam#ab199157), ATXN7 (Invitrogen#PAI-749), CREBBP (CST#7389), SAPK/JNK (CST#9252), LPAR2 (Abcam#ab135980), NF1 (Bethyl#A300-140A-M), Phospho-p38 MAPK (Thr180/Tyr182) (CST#4511), p38 MAPK (D13E1) (CST#8690), p44/42 MAPK (Erk1/2) (CST#4695), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST#4370), Phospho-SAPK/JNK (Thr183/Tyr185) (CST#4668), TRAIL (CST#3219), RIP140 (Santa Cruz#sc518071), GAPDH (Santa Cruz#sc32233), c-Myc (CST#5605), TGFBR2 (Santa Cruz#sc17792), CASZ1 (Santa Cruz#sc398303), and CFL1 (Abcam#ab42824). Full blots are shown in Additional file 1 : Fig. S11-S13.
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