Paraffin-embedded tissue sections were labeled with mouse SOX9 antibody (Abcam) or its isotype control (Vector Biolabs) detected with anti-mouse IgG conjugated to Alexa 488. To detect LOXL2 expression, the tissues were subjected to rabbit anti-LOXL2 (GeneTex), anti-RFP (Abcam), anti-phospho-SMAD2/3 (Cell Signaling Technology), anti-COL2A1 (Abcam) or its isotype control antibody and detected with anti-rabbit Alexa 488-conjugated antibody or biotin antibody followed by streptavidin-conjugated Texas red, in respective samples. Anti-fade reagent with DAPI was added to all samples. A Zeiss 710 dual scanner confocal microscope with a Plan-Apochromat objective, oil immersion lens, and a CCD detector was used to obtain confocal images. Image acquisition was performed with Zeiss Zen image analysis software (Carl Zeiss Micro Imaging). The image analysis was performed using Zeiss LSM viewer and Image J software (NIH). Z-stack images analysis and 3-dimensional reconstruction was performed by using LOCI and the 3D viewer plug-in of Image J software. Quantification was performed using Image J software as described [15 (link)].
Anti phospho smad2 3
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-SMAD2/3 is a laboratory reagent used to detect the phosphorylated forms of the SMAD2 and SMAD3 proteins. SMAD2 and SMAD3 are transcription factors that play a key role in the TGF-beta signaling pathway. This product can be used in various applications such as Western blotting, immunohistochemistry, and flow cytometry to analyze the activation status of the TGF-beta pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti smad2 3, by Cell Signaling Technology (7 mentions)
Anti vimentin, by Cell Signaling Technology (3 mentions)
Anti phospho erk, by Cell Signaling Technology (3 mentions)
Anti rhoa, by Cell Signaling Technology (2 mentions)
Anti cdc42, by Cell Signaling Technology (2 mentions)
Anti pkcε, by Cell Signaling Technology (2 mentions)
Anti e cadherin, by R&D Systems (2 mentions)
Anti pkcε, by Santa Cruz Biotechnology (2 mentions)
Anti phospho mypt1, by Cell Signaling Technology (2 mentions)
Anti pkcδ, by Cell Signaling Technology (2 mentions)
Anti vinculin, by Merck Group (2 mentions)
Anti β actin, by BD (2 mentions)
Anti n cadherin, by Thermo Fisher Scientific (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Anti smad2, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Rabbit anti loxl2, by GeneTex (1 mentions)
Anti rfp, by Abcam (1 mentions)
18 protocols using anti phospho smad2 3
1
Immunohistochemical Analysis of SOX9 and LOXL2
2
Western Blot Analysis of SMAD Signaling
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Cell lysates were prepared using lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% triton X-100) with mini protease inhibitor cocktail tablet (Roche) and phosphatase inhibitor sodium vanadate (2 mM). The following antibodies were used: anti-SMAD 2/3 antibody (#3102S), antiphospho-SMAD 2/3 (#8828S), horseradish peroxidise conjugated secondary antibody (#7074S) from Cell Signalling Technology. The specific protein was detected by treating the membrane for two minutes with enhanced chemiluminescence (# 133406, Abcam) which provides the HRP substrate, and capturing the signal in X-ray films.
3
Western Blot Analysis of Key Signaling Proteins
4
Protein Extraction and Western Blot Analysis
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HOK cells were harvested and then lysed in a lysis buffer supplement with protease inhibitors (Beyotime Institute of Biotechnology, Haimen, China) and phosphatase inhibitors (Nanjing KeyGEN Biotech. Co., Ltd, China) for extraction of total cellular protein. The protein quantification was obtained by bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). For western blot analyses, 20-30 μg of total extracted proteins were injected into each lane before SDS-PAGE. Following transfer to polyvinylidene fluoride membranes, polyclonal anti-YOD1 (Proteintech, USA), anti-TGF-β3 (Proteintech), anti-Smad2/3 (Cell Signaling, Danvers, MA), and anti-phospho-Smad2/3 (Cell Signaling) were used for the detection of the protein expression levels. The expression of beta-tubulin (Cell Signaling) was used as an internal reference for protein loading.
5
Analyzing Signaling Pathways in C2C12 and BRITER Cells
6
Characterization of Cell Lineages
7
Western Blot Analysis of Signaling Pathways
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The cultured cells were washed twice with ice-cold PBS. They were incubated with 2× SDS buffer (100 mM Tris-HCl [pH 6.8], 2% [w/v] SDS, 0.01% bromophenol blue, 20% glycerol, and 10% β-mercaptoethanol) for 5 min at 25 °C. The cells were then collected by scraping with cell scrapers and proteins were denatured at 95 °C for 5 min. Western blot analysis was performed using the following primary antibodies: anti‐phospho‐Smad 2/3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti‐Smad2 (1:1,000; Cell Signaling Technology), anti-N-cadherin (1:2,000, Invitrogen), anti‐phospho-ERKs (1:5,000; Cell Signaling Technology), anti‐ERKs (1:2,000; Cell Signaling Technology), anti‐phospho‐Akt (1:4,000; Cell Signaling Technology), anti‐Akt (1:1,000; Cell Signaling Technology), anti‐phospho‐p38 (1:1,000; Cell Signaling Technology), anti‐p38 (1:1,000; Cell Signaling Technology), anti-Smad4 (1:1,000; Cell Signaling Technology), anti-OB-cadherin (1:1,000; Cell Signaling Technology), and α‐tubulin (1:20,000; Sigma). Densitometry of the bands obtained was performed with the ImageJ software (NIH, Bethesda, MD, USA).
8
Western Blot Analysis of Cell Signaling
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Cells were lysed in a buffer containing 150 mM NaCl, 1.0% nonidet-P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 8.0) and a protease inhibitor cocktail (Roche Applied Science, Vienna, Austria, pH 7.4). Frozen tissue samples stored in liquid nitrogen were minced with scissors. Each sample was homogenized in a lysis buffer at a ratio of 1:20 w/v. After centrifugation at 13,000 rpm for 20 min, the supernatant was used to measure the total protein. Electrophoresis was performed as described previously [48 (link)]. The following primary antibodies were used for Western blot analysis: anti-GDF15 (1:1000; Abcam, Cambridge, UK), anti-phospho-AKT (Ser 473), anti-total AKT, anti-phospho-ERK, anti-total ERK, anti-phospho-SMAD2/3, anti-total SMAD2/3, anti-EGR1, anti-vimentin, anti-Snail, anti-β-actin (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA), anti-N-cadherin, anti-E-cadherin and anti-GAPDH (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA). Following incubation with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology), immune reactive bands were visualized by enhanced chemiluminescence detection (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
9
Quantitative Analysis of Smad2/3 Signaling
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The skin tissues were crushed into pieces using an automill machine (2 sets of 2 min at 1500 rpm) in RIPA buffer supplemented with protease inhibitor: PMSF (0.1 mM) and phosphatase inhibitor: sodium fluoride (5 mM).
The protein concentration was analysed using Qubit protein assay kit (Thermo Fisher Scientific, Cleveland, OH, USA).
Western blotting was performed using the Protein Simple WES System (Protein Simple, SanJose, CA, USA) following the protocols given by the manufacturer. Primary antibodies: anti-Smad2/3 (1:20) (Cell Signaling Technologies, Danvers, MA, USA), anti-Phospho-Smad2/3 (1:20) (Cell Signaling Technologies), and secondary antibodies: Anti-Rabbit Secondary HRP Antibody (Protein Simple) were used the western blot. Protein expression was normalized to total protein using the Total Protein Detection Module (Protein Simple). Simple Western data was analysed using Compass Software version 3.1 (Protein Simple).
The protein concentration was analysed using Qubit protein assay kit (Thermo Fisher Scientific, Cleveland, OH, USA).
Western blotting was performed using the Protein Simple WES System (Protein Simple, SanJose, CA, USA) following the protocols given by the manufacturer. Primary antibodies: anti-Smad2/3 (1:20) (Cell Signaling Technologies, Danvers, MA, USA), anti-Phospho-Smad2/3 (1:20) (Cell Signaling Technologies), and secondary antibodies: Anti-Rabbit Secondary HRP Antibody (Protein Simple) were used the western blot. Protein expression was normalized to total protein using the Total Protein Detection Module (Protein Simple). Simple Western data was analysed using Compass Software version 3.1 (Protein Simple).
10
TGFβ Signaling Pathway Modulation
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Primary cultured iKAP CRC cells were treated with 2 ng/mL recombinant TGFB1 protein or 10 µM TGFβR1 inhibitor galunisertib for 24 h and harvested. Western blotting was performed under standard methods using anti-phospho-SMAD2/3 (Cell Signaling Technology, 8828) and anti-phospho-ERK (Cell Signaling Technology, 4370).
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