RT4 and KMBC2 cells transfected with siNC and siNodal were washed three times with cold PBS and then lysed using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing phenylmethane sulfonyl fluoride. Proteins (40 µg per sample) were separated on 10% sodium dodecyl sulfate–polyacrylamide gels and then transferred electrophoretically onto a PVDF membrane. The membranes were blocked with 5% bovine serum albumin diluted in TBST and then incubated with appropriate antibodies against Nodal, CDC6, E-cadherin, MMP-2, MMP-9, ALK4, ALK7, Smad2, Smad4, and GAPDH (1:1,000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The membranes were then washed three times with TBST, immediately followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1,000; Cell Signaling Technology) for 1 hour at room temperature. GAPDH was used as the internal control. Protein bands were detected using an enhanced chemiluminescence kit (ECL kit, New York, NY, USA) and visualized by autoradiography on an X-ray film.
Smad4
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
Smad4 is a protein that functions as a central mediator in the transforming growth factor-beta (TGF-β) signaling pathway. It is responsible for transmitting signals from the cell surface to the nucleus, playing a crucial role in regulating gene expression in response to TGF-β stimulation.
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Lab products found in correlation
Smad2, by Cell Signaling Technology (32 mentions)
Smad3, by Cell Signaling Technology (23 mentions)
P smad3, by Cell Signaling Technology (16 mentions)
P smad2, by Cell Signaling Technology (16 mentions)
Gapdh, by Cell Signaling Technology (11 mentions)
E cadherin, by Cell Signaling Technology (9 mentions)
Vimentin, by Cell Signaling Technology (9 mentions)
β actin, by Merck Group (9 mentions)
Smad2 3, by Cell Signaling Technology (8 mentions)
N cadherin, by Cell Signaling Technology (8 mentions)
Ripa buffer, by Thermo Fisher Scientific (8 mentions)
β actin, by Cell Signaling Technology (7 mentions)
Phospho smad2, by Cell Signaling Technology (7 mentions)
Snail, by Cell Signaling Technology (7 mentions)
E cadherin, by Abcam (5 mentions)
β actin, by Santa Cruz Biotechnology (5 mentions)
Vimentin, by Abcam (5 mentions)
Phospho smad3, by Cell Signaling Technology (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Erk1 2, by Cell Signaling Technology (4 mentions)
89 protocols using smad4
1
Molecular Profiling of Nodal Signaling
2
Western Blot Characterization of Protein Markers
3
Western Blot Analysis of Protein Targets
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Protein or cell extracts were resolved on a 10% SDS–PAGE gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked with 5% skim milk in TBST for 1 h at room temperature. The membrane was then probed with an antibody against FLAG, Foxp3 (eBioscience), Smad2 (Cell Signaling, 5339), pSmad3 (Cell Signaling, 9520), Smad3 (Abcam, 29379), Smad4 (Cell Signaling, 9515), YY1 (Santa Cruz, sc-1703) or β-actin (Santa Cruz, sc-47778), diluted 1:100 or 1:1,000 in TBST overnight at 4 °C. An HRP-conjugated antibody against rabbit or mouse (BioLegend) diluted at 1:2,000 in 5% skim milk TBST was added for 1 h at room temperature. Target proteins were detected by enhanced chemiluminescence reaction. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 7 .
4
Analyzing TGF-β Signaling Pathway
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Antibodies: smad3, p-smad3, smad2, smad4, smad1, smad5, smad8, p15, p21, p27, p57, c-myc, CDK4, CDK6, β-actin, TBP antibodies were purchased from Cell Signaling Technology. Goat anti-mouse antibody, goat anti-rabbit antibody were also from Cell Signaling Technology. TbRI and TbRII inhibitor LY2109761 was purchased from Selleckchem. Homoharringtonine was purchased from Sigma.
5
Protein Extraction and Western Blotting
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Protein extraction and Western blotting experiments were performed as described previously [12 (link)]. Primary antibodies used were as follow: anti-TGF-β1, CDK4, c-Myc, CyclinD3, p27, phospho-Akt, Akt, phospho-Erk1/2, Erk1/2, phospho-JNK, JNK, phospho-p38, p38, phospho-Smad 2, Smad 2/3, Smad 4, and Histone H3 (Cell Signaling, Danvers, MA); anti-CD63, CD81, and CD9 (Santa Cruz, Santa Cruz, CA); and anti-β-actin (Sigma-Aldrich). HRP-conjugated horse anti-mouse and goat anti-rabbit IgG (Cell Signaling) were used as secondary antibodies.
6
Protein Expression Analysis by Western Blot
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Total proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich) and quantified by Bicin Choninic Acid (BCA) protein assay kit (Thermo). The Western blot was performed according to standard procedures. The following antibodies were used: EphA8 (Abcam, USA), TGFβ1, TGFβR1, smad2, smad3 and smad4 (Cell Signaling Technology, Beverly, MA, USA); β-actin (Proteintech, USA) was used as loading control. The blots were incubated with goat anti-rabbit or anti-mouse secondary antibodies (Bioss, Beijing, China). The proteins were visualized using a chemiluminescence method (ECL Plus Western Blotting Detection System; Amersham Biosciences, Foster City, CA, USA). The bands were quantified by ImageJ software.
7
Western Blot Analysis of Stem Cell Markers
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Cells were processed for western blotting, as described previously (Brouxhon et al., 2007 (link)). Briefly, samples were electrophoretically separated using SDS‐PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with 5% (w/v) nonfat dry milk to block nonspecific binding, and then incubated with TGF‐βRI, TGF‐βRII, Sox2 and β‐actin (Santa Cruz Biotechnology), Oct4 (Chemicon), Nestin (Novus Biologicals), Nanog, TGF‐βRIII, phospho‐specific Smad2, Smad3, Smad2, Smad3, and Smad4 (Cell signaling). Proteins were detected using appropriate HRP‐conjugated secondary antibodies (Santa Cruz Biotechnology). Immunoreactive bands were detected using the Clarity MaxTM Western ECL Substrate (Bio‐Rad), and visualized using the ChemiDoc MP Imaging System (Bio‐Rad). Western blot signals were normalized to β‐actin, using NIH Scion Image. Results are presented as fold increase relative to controls in triplicate experiments.
8
Western Blot Antibody Catalog
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PDE3A (catalog number A302-740A, 1:1000 [Bethyl Laboratory]; catalog number Sc-293446, 1:20 [Santa Cruz Biotechnology]), Flag (catalog number F7425, 1:2000; Sigma), GAPDH (catalog number 60004-1-1g, 1:5000; ProteinTech), Anti-phospho-PKA substrate antibody (catalog number 9621S or 9624, 1:1000; Cell Signaling), SMAD4 (catalog number 46535, GTX01674; Cell Signaling) GATA4 (GATA4 binding protein 4; catalog number 36966, 1:1000; Cell Signaling), HDAC-1 (catalog number 34589, 1:1000; Cell Signaling), lamin B (catalog number 12586, 1:1000; Cell Signaling), lamin A/C (catalog number 4777S, 1:1000; Cell Signaling), actin-1 alpha (catalog number 17521-1-AP; Proteintech), RFP (catalog number ab62341, 1:2000; Abcam), GFP (catalog number ab6556, 1:1000; Abcam), horseradish peroxidase–conjugated goat anti-mouse (catalog number sc2005, 1:5000 [SCBT]; catalog number 705-035-151 [Jackson ImmunoResearch Labs]) or anti-rabbit secondary antibodies (catalog number sc2030, 1:5000 [Santa Cruz Biotechnology]; catalog number 705-035-152 [Jackson ImmunoResearch Labs]), antibodies conjugated with Alexa-Fluor 647 (catalog number A-21235, 1:250; Thermo Fisher Scientific), Alexa-Fluor 488 (catalog number A-11008, 1:250; Thermo Fisher Scientific), and wheat germ agglutinin conjugated with Alexa 488 (catalog number W11261; Invitrogen).
9
Protein Extraction and Western Blot Analysis
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We extracted total protein from the cell lines and tissues using radioimmunoprecipitation buffer (Sigma-Aldrich Co.) and determined the protein concentration using the BCA Protein Assay Kit (Beyotime, Shanghai, China). The extraction of nuclear protein extractions were prepared using an extraction kit (Beyotime) according to the manufacturer’s protocols as reported previously.16 (link) The proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking in 5% BSA for 1 hour, the membranes were incubated overnight at 4°C with β-actin (1:2,000; Cell Signaling Technology, Danvers, MA, USA), INTS8 (1:200; Abcam), N-cadherin (1:1,000; Abcam), E-cadherin (1:1,000; Abcam), Vimentin (1:1,000; Abcam), Histone H3 (1:2,000; Cell Signaling Technology), and SMAD4 (1:1,000; Cell Signaling Technology) antibodies. The secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000) was then applied for 1 hour at 37°C. β-actin served as the internal control for all WBs. Histone H3 served as the internal control for nuclear protein. We quantified protein expression using Bio-Rad Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA).
10
Lung Fibroblast Cell Culture Protocol
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