These antibodies were used in the present investigation: Kindlin-1 (Millipore, MA), c-myc (Snail (Millipore), E-cadherin (Abcam), N-cadherin (Epitomics), p-Smad3 (Abcam), Smad2 (Epitomics), Smad3 (Epitomics), SARA (Epitomics), TβRI (Santa Cruz, CA), TβRII (Santa Cruz, CA), Vimentin (Epitomics), YY-1 (Santa Cruz, CA), Actin (Santa Cruz), or Flag (Sigma-Aldrich, St. Louis, MO).
Smad2
Manufactured by Abcam
Sourced in United States, United Kingdom, Germany
Smad2 is a cellular protein that functions as a signal transducer and transcriptional modulator. It is a key component of the TGF-beta signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
P smad2, by Abcam (20 mentions)
Smad3, by Abcam (15 mentions)
Tgf β1, by Abcam (10 mentions)
P smad3, by Abcam (9 mentions)
E cadherin, by Abcam (7 mentions)
β actin, by Abcam (7 mentions)
Gapdh, by Abcam (5 mentions)
Vimentin, by Abcam (4 mentions)
α sma, by Abcam (4 mentions)
β actin, by Merck Group (3 mentions)
Phospho smad2, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Smad3, by Cell Signaling Technology (3 mentions)
P smad2, by Cell Signaling Technology (3 mentions)
N cadherin, by Abcam (2 mentions)
E cadherin, by BD (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Vimentin, by Merck Group (2 mentions)
Phospho smad3, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
45 protocols using smad2
1
Antibody Validation for Cell Signaling
2
TGF-β1 Signaling Pathway Analysis
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The following antibodies were used: ALK5 (V-22), HSP90α/β (H-114, sc-7947), and PAR2 (SAM11, sc-13504, Fluorescein isothiocyanate (FITC)-conjugated) (Santa Cruz Biotechnology, Heidelberg, Germany), Caveolin (BD Transduction Laboratories, Heidelberg, Germany, #610059), phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-Smad2(Ser465/467) (all from Cell Signaling Technology, Frankfurt, Germany, #4370, #3104 and #2276, respectively), phospho-Smad3(Ser423/425) (R&D Systems, Wiesbaden, Germany, #ab3226), Smad2 (Epitomics, Burlingame, CA, #1736-1), Smad3 (Abcam, Cambridge, UK, #ab40854), TGF-β Receptor II (Cell Signaling Technology, #11888), β-actin (Sigma, Deisenhofen, Germany). TGF-β1 was purchased from either R&D Systems or ReliaTech (Wolfenbüttel, Germany) and SB431542 [42 (link)] from Sigma. Pharmacological inhibitors were added to cells 30-60 min before the addition of TGF-β1 which was used at a concentration of 5 ng/ml.
3
Western Blot Procedure for Protein Analysis
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The Western blot procedure was performed as described in detail earlier [19 (link)]. Briefly, equal amounts of crude proteinaceous extracts from each cell line (three preparations harvested at different times during the continuous culture) were fractionated by SDS-PAGE on mini-PROTEAN TGX any-kD precast gels or TGX Stain-Free FastCast gels (BioRad, Munich, Germany) and blotted to PVDF membranes. The membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies to E-cadherin (#610181, BD Transduction Laboratories, Heidelberg, Germany) or vimentin (clone V9, #V6630, Sigma-Aldrich, Steinheim, Germany), phospho-Smad2 (S465/S467) (#3101, Cell Signaling Technology, Frankfurt/Main, Germany), Smad2 (#1736-1, Epitomics, Burlingame, CA, USA), and GAPDH (Cell Signaling Technology) as loading controls.
4
Western Blot Analysis of Smad Signaling
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Inhibitor-treated cells were lysed in PhosphoSafe buffer (Merck) and the protein concentrations determined with the DC protein assay (Bio-Rad, München, Germany). Equal amounts of cellular proteins were fractionated by SDS-PAGE, transferred to PVDF membrane and immunoblotted as described in detail earlier [26 (link)]. The antibodies used were: β-actin (Sigma-Aldrich, #A1978), N-cadherin (BD Transduction Lab. #610920), anti-phospho-Smad2(Ser465/467) and anti-phospho-Smad3(Ser423/425), both from Cell Signalling Technology (Frankfurt/Main, Germany), Smad2 (Epitomics, Burlingame, CA, #1736-1), Smad3 (Abcam, Cambridge, UK, #ab40854), Snail (Cell Signalling Technology, #4719), and vimentin (Sigma-Aldrich, #V6630). In some experiments, the intensities of bands were quantified by densitometry using NIH image J.
5
Western Blot and IHC Analysis of Tumor Proteins
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Protein lysates of tumour tissues or cultured cells were prepared using RIPA buffer (Sigma-Aldrich; Merck KGaA) containing 1% phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitors. Western blot analysis was performed as previously described (11 (link)). The antibodies used were as follows: MMP11 (product code ab119284; 1:1,000 dilution; Abcam), Smad2 (cat. no. 12570-1-AP; 1:1,000 dilution), Smad3 (cat. no. 25494-1-AP; 1:1,000 dilution), and β-actin (cat. no. 20536-1-AP; 1:1,000 dilution) primary antibodies and goat anti-rabbit IgG secondary antibody (cat. no. SA00001-2; 1:5,000 dilution; all from ProteinTech Group, Inc.). For immunohistochemistry (IHC), the aforementioned antibodies MMP11 and Smad2 (1:1,000 dilution) were used. The specific immunohistochemistry protocol was performed as previously described (12 (link)).
6
Inflammatory Cytokine Regulation Analysis
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Lipopolysaccharide was purchased from Sigma-Aldrich (St Louis, Ca). Recombinant mouse IL-4, recombinant mouse IL-6, recombinant mouse IL-1β, recombinant mouse TNF-α, recombinant mouse IL-10, and recombinant mouse TGF-β were purchased from R&D Systems (Minneapolis, MN). Tocilizumab, LY2109761, R-7050, Stattic, Bay 11-7082, and SIS3HCl were purchased from Selleck (Huston, USA). IL-1 receptor antagonist (IL1Ra) was purchased from Make Research Easy (Nanjing, China). Monoclonal mouse anti-β-actin (TA-09) was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Antibodies against OT, OTR, β-tubulin, CD206, F4-80, phospho-STAT3, STAT3, phospho-SMAD2, SMAD2, phospho-SMAD3, SMAD3, p65, and phospho-p65 were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Danvers, MA). Secondary antibodies were purchased from Invitrogen Life Technology (Foster City, CA). Mouse ELISA kits were obtained from R&D Systems and CUSABIO (Wuhan, China). All reagents were analytical grade.
7
Protein Expression Analysis by Western Blot
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The entire protein lysates were split by 10% SDS‐PAGE. The split protein was transferred into a PVDF membrane (Millipore, Bedford, MA, USA). The above membranes were incubated with the primary Abs SMAD2 (1:1000 dilution; Abcam, Cambridge, UK) and GAPDH (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight. The membranes were then treated with secondary Ab conjugated to HRP for 2 hours at room temperature. Detection was undertaken using an ECL western blotting kit (Amersham Biosciences, Little Chalfont, UK). GAPDH was used as the loading control. The intensities of protein bands were quantitated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
8
Exosomal lncRNA Inhibition Impacts HUVEC Signaling
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HUVECs were seeded to 6 well plates (2×105 cells per well) and treated with ovarian cancer-derived exosomes with or without lncRNA siRNA2. Then, HUVECs were collected in RIPA lysis buffer (ASPEN Biotechnology CO., LTD, Wuhan, China) and was lysed in the culture plate for 5 min. Next, the protein concentrations were quantified by The BCA protein assay kit (Beyotime, Shanghai, China). Proteins samples (30 μg/lane) were separated by 10% SDS–PAGE, followed by transferred onto PVDF membranes (Millipore). After that, the membranes were incubated with primary antibodies of CD81 (1:1000, Abcam, Cambridge, MA, USA, cat. no. ab109201), TSG101 (1:1000, Abcam, cat. no. ab125011), p-Smad2 (1:1000, Abcam, cat. no. ab280888), Smad2 (1:1000, Abcam, cat. no. ab33875), E-cadherin (1:1000, Abcam, cat. no. ab231303), N-cadherin (1:1000, Abcam, cat. no. ab76011) and β-actin (1:1000, Abcam, cat. no. ab8226) at 4°C overnight. On the following day, the membrane was washed with TBST for three times and incubated with corresponding secondary antibodies for 1 h at room temperature. Finally, an enhanced chemiluminescence (ECL) reagent (Millipore) was used to visualize the bands.
9
Western Blot Analysis of TGF-β Signaling Proteins
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BFTC-909 cells were lysed in RIPA buffer containing a protease inhibitor mixture (Roche, Basel, Switzerland). For each lane of 10% SDS–PAGE gel, 30 μg of cell lysate protein was loaded, separated, and subsequently transferred onto Immobilon-P Transfer Membrane (Millipore, Burlington, MA, USA). The membranes were probed with specific antibodies including the primary antibodies against INHBA (cat# sc-166503; Santa Cruz, CA, USA; 1:500), SMAD4 (cat# sc-7966; Santa Cruz, CA, USA; 1:1000), SMAD3 (cat# ab40854; Abcam, Cambridge, UK; 1:1000), SMAD2 (cat# ab40855; Abcam, Cambridge, UK; 1:1000), and beta-actin (cat# ZRB1312; Sigma, St. Louis, MO, USA; 1:5000). The secondary antibodies were added and incubated for 1 h and visualized using chemiluminescence. Enhanced chemiluminescence Western blotting reagents were obtained from Pierce Biotechnology (Rockford, IL, USA).
10
Comprehensive Analysis of Tumor Microenvironment
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YFTL was obtained from Changsha Central Hospital (Hunan, China). The HE staining kit was purchased from Beyotime (Jiangsu, China). Antibodies against proliferating cell nuclear antigen (PCNA), p53, MMP-2, MMP-9, E-cadherin, N-cadherin vimentin, VEGF, TGFβ1, Smad2, and p-Smad2 were purchased from Abcam (Cambridge, UK). The cytokine ELISA kits were purchased from Lianke Biotechnology Co. Ltd. (Hangzhou, China). Anti-CD3+ (FITC), anti-CD4+ (PE-Cy5), anti-CD8+ (PE) and anti-NK1.1(PE-Cy7) antibodies were provided by Bio Legend, Inc. (San Diego, CA, USA). Anti-AKT and anti-p-AKT antibodies were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against ERK1/2, p-ERK1/2, p38, p-p38, JNK, and p-JNK were from Cell Signaling Technologies (Danvers, MA, USA). Anti-β-actin antibodies were from Proteintech Biotechnology (Rocky Hill, USA). Other reagents were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).
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