B16-F10 cells were co-cultured with DLNC or Tregs at various co-culture ratios for 72 h. Western blotting was performed as described previously [31 (link)]. Blocked membranes were incubated with primary Abs against Foxp3 (cat. no. ab54501, abcam, Cambridge, MA, USA), TGF-β (cat. no. ab9758, abcam), Smad2/3 (cat. no. 8685, clone D7G7, Cell signaling technology, Beverly, MA), β-catenin (cat. no. 9587, Cell signaling technology), α-SMA (alpha-smooth muscle actin; cat. no. ab5694, abcam), vimentin (cat. no. 3932, clone R28, Cell signaling technology), or MMP9 (Matrix metalloproteinase 9; cat. no. ab137867, clone EP1255Y, abcam) overnight at 4 °C. The blots were incubated with the following secondary Abs conjugated to horseradish peroxidase: goat anti-rabbit IgG (cat. no. 7074, Cell signaling technology), goat anti-mouse IgG (cat. no. 7076, Cell signaling technology), or mouse anti-goat IgG (cat. no. 14-13-06, KPL/SeraCare, Gaithersburg, MD, USA) and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Uppsala, Sweden). Protein expression was semi-quantitatively analyzed using ImageJ software (version 1.50b; U.S. National Institutes of Health, Bethesda, MD, USA).
Smad2 3
Manufactured by Abcam
Sourced in United States, United Kingdom
Smad2/3 is a protein complex that plays a central role in the transforming growth factor-beta (TGF-β) signaling pathway. It functions as an intracellular signal transducer and transcriptional modulator, regulating the expression of target genes in response to TGF-β family ligand activation.
Automatically generated - may contain errors
Lab products found in correlation
P smad2 3, by Abcam (10 mentions)
Tgf β1, by Abcam (6 mentions)
α sma, by Abcam (6 mentions)
Vimentin, by Abcam (3 mentions)
Tgf β, by Abcam (2 mentions)
Smad4, by Abcam (2 mentions)
P akt, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
β actin, by Proteintech (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
α sma, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Odyssey two color infrared laser imaging system, by LI COR (1 mentions)
Sa5 35521, by Thermo Fisher Scientific (1 mentions)
Notch3, by Proteintech (1 mentions)
Bcl2 associated x (bax), by Abmart (1 mentions)
17 protocols using smad2 3
1
Western Blot Analysis of Immune Cell Interactions
2
Protein Expression Analysis in Cells
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RIPA buffer (Solarbio, Shanghai, China) was used to lyse cells and a BCA kit (Beyotime, Shanghai, China) was used to quantify protein levels. The protein concentration was about 50 mg/mL, which was separated by 10% SDS-PAGE gel. β-actin (1:5000, AbMART, Shanghai, China, M2009) was used as a loading control. The antibody Fam98b (1:2000, FineTest, Wuhan, China, FNab03001), α-SMA (1:2000, Abcam, Cambridge, UK, ab124964), Collagen I (1:2000, Abcam, Cambridge, UK, sc-59772), Smad2/3 (1:2000, Abcam, Cambridge, UK, ab63672), BAD (1:2000, AbMART, Shanghai, China, 67830-1-Ig), BAX (1:2000, AbMART, Shanghai, China, T40051), Bcl2 (1:2000, AbMART, Shanghai, China, T40056), TGFβ (1:500, Wanleibio, Shenyang, China, WL02998), P-Smad2/3 (1:1000, Absin, Shanghai, China, abs131873), NOTCH3 (1:1000, Proteintech, Wuahn, China, 55114-1-AP) were used as primary antibody. The secondary antibodies were obtained from Invitrogen (1:20,000, Thermofisher, SA5-35521, SA5-35571). An Odyssey two-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA) was employed to scan the blots. The quantitative analysis of grey values was performed using Image J software (NIH, USA).
3
Western Blot Analysis of Signaling Proteins
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Protein lysates were homogenized by RIPA buffer with phosphatase and protease inhibitors. Proteins (30 µg) were separated in 4–15% polyacrylamide gel and then transferred to PVDF membrane (0.2 mM) by Trans-Blot Turbo (BioRad, Hercules, CA, USA). After blocking in BSA at 5%, the membranes were incubated overnight with the following primary antibodies: pSMAD2/3 (Abcam), SMAD 2/3, pERK, extracellular signal-regulated kinases (ERK), Id2, matrix metallopeptidase (MMP9) (Santa Cruz Biotechnology, Inc.) and then with secondary antibody (hrp-conjugated, Santa Cruz). The same membrane was probed with mouse monoclonal anti-βactin antibody (1:20,000; Sigma). The electrochemiluminescence system was used to detect the antibody binding (Amersham, UK). The chemiluminescent signal was acquired by Chemidoc and quantified using Image J software.
4
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).
5
Hepatic Fibrosis Regulation by TGF-β1
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The TGF-β1 antibody was purchased from R&D Systems (Minneapolis, MN, United States); antibodies against GLUT1, p-Smad2/3, Smad2/3, p-P38, p-AKT and desmin were purchased from Abcam (Cambridge, MA, United States), and the tubulin antibody was purchased from Research Diagnostics (Flanders, NJ, United States). The anti-α-SMA antibody, carbon tetrachloride (CCl4), corn oil, OptiPrep and other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, United States) and Fisher Scientific (Waltham, MA, United States). A TβRI/II inhibitor (APExBIO Technology, United States) was used at 2 μmol/L. Inhibitors of p38 MAPK and PI3K, namely, SB203580 and LY294002, respectively, were purchased from Abcam (Cambridge, MA, United States). The Smad3 inhibitors SIS3 and phloretin were purchased from Abcam (Cambridge, MA, United States).
6
Immunohistochemical Analysis of TGF-β Signaling
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Slices were dewaxed in xylene and rehydrated in ethanol grading. After rehydration, slices were quenched in 0.3% hydrogen peroxide in methanol. The rehydrated sections were subjected to antigen retrieval in gastric enzyme for 30 minutes followed by incubation with primary antibodies. The slides were incubated at 4 ° overnight with rabbit antibodies against TGF-β (Abcam, Cambridge, MA), type II collagen (Abcam, Cambridge, MA), Smad2/3 (Abcam, Cambridge, MA), Smad1/5/8 (Novus Biologicals, Inc., USA) at dilutions of 1:100, 1:100, 1:500 and 1:400 respectively, and then incubated with secondary antibodies (ZSGB-BIO, Beijing, China) at room temperature for 30 min. DAB (ZSGB-BIO, Beijing, China) was used to visualize the color. Images were captured by Image-Pro Plus using a digital slide scanner (Hamamatsu, Japan). The mean integrated optical density (IOD) was used to present the positive intensity of immunohistochemical expression. Three sections per sample were analyzed.
7
Western Blot Analysis of EMT Markers
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Cells were lysed in RIPA buffer (SolarBio, 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% (v/v) NP-40, 0.1% (w/v) SDS) containing 1% (v/v) phenylmethylsulfonyl fluoride (SolarBio), 0.3% (v/v) protease inhibitor (Sigma Aldrich), and 0.1% (v/v) phosphorylated proteinase inhibitor (Sigma) and then clarified by centrifugation. The supernatant was collected and then separated on an SDS-PAGE gel (10% (v/v) polyacrylamide), transferred onto a PVDF membrane. Nonspecific binding was blocked in Tris-buffered saline with Tween 20 (TBS-T) with 8% (w/v) milk for 2 h. After incubation with primary antibodies against β-actin (Abmart), E-cadherin (E-Cad, Abcam), vimentin (Vi, Abcam), α-smooth muscle actin (α-SMA, Sigma), Smad2/3 (Abcam), and p-Smad2/3 (Abcam) for overnight at 4 °C, the membranes were washed with TBS-T for several times and incubated in HRP-linked secondary antibodies (Abmart) for 2 h at room temperature (RT). After repeated washing with TBS-T, the immunoreactive proteins were visualized using enhanced chemiluminescence (Millipore) according to the manufacturer’s instructions and quantified using density analysis, normalized against β-actin and expressed as the fold change compared with the control.
8
Western Blot Analysis of Epithelial Markers
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16HBE14o- and RTE cells incubated for different time periods were collected and lysed. The lysates were centrifuged, denatured, applied to SDS polyacrylamide gels for electrophoresis and transferred to polyvinylidene fluoride membranes. Then, the membranes were blocked with 5% skimmed milk at room temperature for 1 h, and immunoblotting was performed using antibodies against NF-κB p65 (Abcam, MA, United States), NF-κB p-p65 (phospho S536, Abcam), p38 (Abcam), p-p38 (phospho Y182, Abcam), α-SMA (Abcam), vimentin (Abcam), E-cadherin (Abcam), ZO-1 (Abcam), Smad2/3 (Abcam), p-Smad2/3 (Abcam), integrin avβ6 (Abcam), β-actin (Abcam) and a TGF-β1-neutralizing antibody (Sigma). After washing, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The bands were visualized via chemiluminescence using an ECL kit (Thermo Scientific) and photographed with a Tanon Multi-Imager. ImageJ (Scion Corporation, Frederick, MD, United States) was used to measure the density of the immunoreactive bands, which was normalized to that of β-actin.
9
Protein Extraction and Western Blot Analysis
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Radioimmunoprecipitation buffer (Beyotime) was used to extract total protein from GC tissues and cell lines. Protein concentrations were quantified with a BCA Protein Assay Kit (Thermo Fisher Scientific). The primary antibodies used in this study included an antibody against C‐E‐Cad (1:1000; This study), E‐Cad (1:1000; Abcam), N‐Cad (1:1000; AbcamK), Snail (1:1000; Abcam), Slug (1:1000; Abcam), Vimentin (1:1000; Abcam), p‐AKTS473 (1:1000; Abcam), p‐AKTT308 (1:1000; Abcam), p‐PDK1 (1:1000; Abcam), AKT (1:1000; Abcam), PDK1 (1:1000; Abcam), p‐Smad2/3 (1:1000; Abcam), Smad2/3 (1:1000; Abcam). β‐actin (1:5000; Sigma‐Aldrich) was used as a control.
10
Penile Protein Expression Assessment
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Penile tissues or cultured cells were extracted in ice-cold lysis buffer. Protein (50 μg) were loaded on 12% sodium dodecyl sulfate/polyacrylamide (SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA for 2 hours and incubated with the following primary antibodies: TERT (1:1000, Affinity), VEGF (1:1000, ProteinTech), bFGF (1:1000, Affinity), IGF-1 (1:1000, ProteinTech), TGF-β1 (1:1000, Abcam, Cambridge, UK), Smad2/3 (1:1000, Abcam), phospho-Smad2/3 (p-Smad2/3, 1:1000, Abcam), Bcl-2 (1:1000, Affinity), Bax (1:1000, ProteinTech), eNOS (1:1000, Abcam), p-eNOS (1:1000, CST, Danvers, USA), and nNOS (1:1000, Abcam). Membranes were washed and incubated with the appropriate secondary antibodies (1:5000) for 1 hour at room temperature. Protein bands were detected using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Rockford, USA).
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