Samples were solubilized with an equal volume of loading buffer (125 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 0.05% bromophenolblue, 5% β-mercaptoethanol) and were boiled for 10 minutes, then samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transferring to polyvinylidene difluoride membranes and detecting by immunoblotting with primary antibodies against E-cadherin (1:500, Abcam), vimentin (1:500, Abcam), p-Smad2 (1:500, Abcam), p-Smad3 (1:500, Abcam), Smad4 (1:500, Abcam), and Snail (1:500, Abcam), respectively at 4°C overnight. Then HRP-conjugated secondary antibody (CST, Boston, MA) was incubated for 1 hour at room temperature, and visualized by using ECL detection kit (CST) [3 (link)]. β-Actin (1:1,000, Abcam) was used as an internal control.
Smad4
Manufactured by Abcam
Sourced in United States, United Kingdom
SMAD4 is a protein that functions as a key component of the transforming growth factor-beta (TGF-β) signaling pathway. It acts as a transcriptional regulator, mediating the cellular response to TGF-β signaling.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Tgf β1, by Abcam (4 mentions)
Smad3, by Abcam (4 mentions)
P smad3, by Abcam (3 mentions)
P smad2 3, by Abcam (3 mentions)
Smad7, by Abcam (3 mentions)
E cadherin, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
P smad2, by Abcam (2 mentions)
Vimentin, by Abcam (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Fibronectin, by Abcam (2 mentions)
Smad2, by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
P akt, by Abcam (2 mentions)
Smad2 3, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
27 protocols using smad4
1
Evaluating Epithelial-Mesenchymal Transition
2
Western Blot Protein Quantification
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Total cellular protein was extracted 48 h after transfection using protease‐containing RIPA lysis and phosphatase inhibitors. Proteins were then separated by electrophoresis using sodium dodecyl sulfate‐containing polyacrylamide gels and then transferred to polyvinylidene fluoride membranes. The membrane was blocked with 5% skimmed milk and incubated with primary antibodies (SMAD4 and glyceraldehyde 3‐phosphate dehydrogenase; Abcam) and horseradish peroxidase‐conjugated secondary antibody. The blots were visualized using an ECL Western blot kit (Millipore), following the manufacturers' protocol. The image was used for quantitative analysis of western blot bands obtained.
3
Western Blot Analysis of Smad4 Protein
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The cultures were washed twice with PBS and incubated in cell lysis buffer (RIPA; Thermo Fisher Scientific, Inc., Waltham, MA, USA) on ice for 30 min. All cell lysates were centrifuged at the speed of 12,000 rpm for 10 min at 4°C. The concentration of proteins in the supernatant was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Protein extracts were boiled and resolved by 10% polyacrylamide gel by electrophoresis, and further electroblotted onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% BSA for 2 h at room temperature, the PVDF membranes were incubated overnight at 4°C with the following primary antibodies: β-actin (1:1,000; #4970; Cell Signaling Technology, Inc., Danvers, MA, USA), and Smad4 (1:5,000; ab40759; Abcam). The PVDF membranes were then washed with Tris-buffered saline containing 1% Tween-20 (TBST) and probed with the secondary antibody conjugated to horseradish peroxidase (anti-rabbit IgG; 1:1,000; #7074s; Cell Signaling Technology, Inc.) for 2 h at room temperature. Following 3 washes with TBST, detection was performed using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology, Haimen, China) plus the Syngene Bio Imaging system (Syngene, Frederick, MD, USA).
4
Modulation of Extracellular Matrix in Fibrosis
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High glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Product code: 01–052-1), fetal bovine serum (FBS, Product code: 04–400-1), penicillin and streptomycin (Product code: 03–031-1) were purchased from HyClone (Logan, UT, USA). The cell counting kit (CCK8) was obtained from Boster (Wuhan, China; Product code: CK04). Marein was purchased from ChromDex (Irvine, CA, USA; Product code: ASB-00013126-005), antibodies against β-actin (Product code: ab8226), GAPDH (Product code: ab8245), Collagen IV (Product code: ab6586), fibronectin (FN, Product code: ab2413), TGF-β1 (Product code: ab92486), MCP-1 (Product code: ab7202), Smad4 (Product code: ab40759), NF-κB P-65 (Product code: ab16502), and ammonium pyrrolidine dithiocarbamate (PDTC, Product code: ab141406) were from Abcam (Cambridge, MA, USA). AMPK (Product code: 5832 T), p-AMPK (Product code: 2535 T), Smad2 (Product code: 8685 T), p-Smad2 (Product code: 8828 T), Smad3 (Product code: 9523 T), p-Smad3 (Product code: 9520 T), and 5-amino-4-imidazole carboxamide (AICAR) were from Cell Signaling (Danvers, MA, USA, Product code: 9944P). Dorsomorphin (Dor) was obtained from Sigma-Aldrich (St. Louis, MO, USA, Product code: P5499).
5
Comprehensive Immunohistochemical Analysis of Tumor Microenvironment
6
Western Blot Analysis of Cell Signaling
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The specific procedure in our published paper was followed.39 (link) Brief description was as follows: cells at 5 × 105 per well (six-well plate) were lysed as one amount of a sample. Equal amounts of protein extracts were separated on 10% SDS-polyacrylamide gel electrophoresis, and then transferred into a PVDF membrane (Millipore, Germany) at 200 mA for 2 h. PVDF membranes were blotted with 5% milk for 1 h and then the blots were probed overnight with primary antibodies (β-actin, 1:2 000, sc-47778; Cx43, 1:3 000, #11370; N-cadherin, 1:2000, #76057; Smad3, 1:3 000, #28379; Smad4, 1:5000, #40759; p-Smad3, 1:2 000, #52903; Abcam, Cambridge, UK), then added corresponding secondary antibodies (m-IgGКBP-HRP, 1:4 000, sc-516102; mouse antirabbit IgG-HRP, 1:2 000, sc-2357, Santa Cruz) and incubated for 2 h. Signals from PVDF membranes were taken by a Western Blotting Luminol Reagent Kit (Santa Cruz).
7
Smad4 Expression Analysis in SMSCs
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SMSCs were harvested and washed with precooled PBS. Cells were lysed with the RIPA buffer (50 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1% nonidet-P40, 1% sodium deoxycholate, and 0.1% SDS). The protein concentration was determined with the BCA kit (Thermo Scientific). Thirty micrograms of protein was loaded in each lane of the SDS/PAGE (10% gel) (Bio–Rad) and then transferred on to the PVDF membrane. The membrane was blocked with 5% BSA for 1 h at room temperature and then incubated with the indicated primary antibodies against Smad4 (Abcam) and β-actin (Santa Cruz). The membrane was then incubated with HRP–conjugated secondary antibodies for 1 h at room temperature. The band was visualized by putting ECL at the top and bottom.
8
DMY-Mediated Regulation of SMAD/Cyclin Signaling
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The following commercial reagents were used: DMY (>99% purity; Beijing Century Aoke Biotechnology Co., Ltd.), fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), DMEM (Gibco; Thermo Fisher Scientific, Inc.), 0.25% trypsin with 0.02% EDTA (Gibco; Thermo Fisher Scientific, Inc.), MTT reagent (Gibco; Thermo Fisher Scientific, Inc.), antibodies against GAPDH (rabbit anti-human polyclonal; cat. no. E12-052; EnoGene Biotech Co., Ltd.), SMAD3 (rabbit anti-human monoclonal; cat. no. ab40854; Abcam), p-SMAD3 (rabbit anti-human monoclonal; cat. no. ab52903; Abcam), SMAD4 (rabbit anti-human monoclonal; cat. no. ab40759; Abcam), Cyclin A1 (rabbit anti-human polyclonal; cat. no. E1A5313; EnoGene Biotech Co., Ltd.) and Cyclin D1 (rabbit anti-human monoclonal; cat. no. ab134175; Abcam), and a secondary antibody (peroxidase-conjugated AffiniPure goat anti-rabbit IgG; cat. no. ab6721; Abcam).
9
Hepatoprotective Mechanisms of TAA and Silymarin
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TAA and silymarin were obtained from Sigma Aldrich (St. Louis, Missouri, USA). The ELISA kits used to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and r-glutamyl transferase (r-GPT) were acquired from Abcam (Cambridge, UK). Malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) assay kits were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Antibodies against collagen-1, TGF-β1, α-SMA, vimentin, Smad2/3, p-Smad2/3, Smad4, Smad7, p-PTEN, PTEN, p-Akt, Akt, p-PI3K, PI3K, and β-actin were purchased from Abcam (Cambridge, Massachusetts, USA) and Cell Signaling Technology (Danvers, Massachusetts, USA). Anti-mouse and anti-rabbit IgG and HRP-linked-conjugated secondary antibodies were procured from Santa Cruz Biotech. (Santa Cruz, California, USA).
10
Western Blot Analysis of EMT Markers
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Total proteins were extracted from HCC tissues and cells, and their concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Western blotting assay was conducted using specific primary antibodies against Snail, Slug (1 : 1000; Santa Cruz Biotechnology, Dallas, TX, USA), E-Cadherin, Akt, phosphorylated Akt (p-Akt) (1 : 1000; Epitomics, Burlingame, CA, USA), N-Cadherin (Thermo Fisher Scientific), Smad2, p-Smad2, Smad3, Smad4 and GAPDH (1 : 1000; Abcam) and appropriate horseradish peroxidase-conjugated secondary antibodies (1 : 5000; Thermo Fisher Scientific). Protein bands were visualised using an enhanced chemiluminescence detection system (Thermo Fisher Scientific). The average grey scale intensities were measured by IPP v6.0 software (Media Cybernetics Inc.).
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