For this study, we used antibodies to PML (1:250 for IB, 1:100 for IP, sc-966, Santa Cruz Biotechnology, Delaware Ave., Santa Cruz, CA, USA), PML (1:500 for immunohistochemistry (IHC), sc5621, Santa Cruz Biotechnology, Delaware Ave.), HA (1:1000 for IB, 1:200 for IF, A190-108 A, Bethyl Laboratories Inc., Montgomery, TX, USA), β-actin (1:5000 for IB, A5441, Sigma-Aldrich, St Louis, MO, USA), Exportin-1 (CRM1; 1:5000 for IB, 1:2000 for IHC, A300-469A, Bethyl Laboratories Inc.), E-Cadherin (1:500 for IF, 610181, BD Transduction Laboratories, San Jose, CA, USA), EMT antibody Sampler Kit (9782, Cell Signaling Technology, Danvers, MA, USA), which contains rabbit antibodies each used at 1:300 (IB), N-Cadherin and Vimentin antibodies were used at 1:500 for IF, Phospho-Smad2/Smad3 (1:300 for IB, 8828, Cell Signaling Technology), Phospho-Smad2/3 (1:100 for IHC, sc-11769, Santa Cruz Biotechnology), Anti-rabbit IgG, HRP-linked Antibody (1:1000 for IB, 7074, Cell Signaling Technology), Anti-TGFβ Receptor I (1:1000 for IB, 3712, Cell Signaling Technology), Anti-SMAD2 (1:1000 for IB, 3122, Cell Signaling Technology), Anti-SMAD3 (1:1000 for IB, 9513, Cell Signaling Technology), Anti-phospho-SMAD2 (1:500, SAB4504207, Sigma-Aldrich), Anti-phospho-SMAD3 (1:500, SAB4300253, Sigma-Aldrich), Anti-mouse IgG, HRP-linked antibody (1:1000 IB, 7076, Cell Signaling Technology).
Phospho smad2 smad3
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-Smad2/Smad3 is a laboratory product that detects the phosphorylated forms of Smad2 and Smad3 proteins. Smad2 and Smad3 are key intracellular signal transducers for the transforming growth factor-beta (TGF-β) signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Smad2 3, by Cell Signaling Technology (2 mentions)
Smad2 smad3, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Emt antibody sampler kit, by Cell Signaling Technology (1 mentions)
Sc 5621, by Santa Cruz Biotechnology (1 mentions)
Phospho smad2 3, by Santa Cruz Biotechnology (1 mentions)
Sc 11769, by Santa Cruz Biotechnology (1 mentions)
Anti tgfβ receptor 1, by Cell Signaling Technology (1 mentions)
Anti phospho smad2, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
Sc 966, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Anti smad2, by Cell Signaling Technology (1 mentions)
Anti smad3, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Ab8878, by Abcam (1 mentions)
Ab228551, by Abcam (1 mentions)
Ab279290, by Abcam (1 mentions)
Anti mouse alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
5 protocols using phospho smad2 smad3
1
Immunoblotting and Immunoprecipitation Antibodies
2
Immunostaining of Cellular Markers
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The following antibodies were applied: Tetraspanin-4 (NBP1-59438, Novus); RPE 65 (MA1-16578; Thermo Fisher Scientific); GFAP (ab279290, Abcam); CD11b (ab8878, Abcam); integrin α5 (ab288767, Abcam); EOGT (ab190693, Abcam); NDST1 (26203-1-AP, Proteintech); Alix (2171, Cell Signaling); Alix (92,880, Cell Signaling); Smad2/3 (3102, Cell Signaling); Phospho-Smad2/Smad3 (8828, Cell Signaling); Alpha-Smooth Muscle Actin (MA5-11547, Thermo Fisher Scientific); GAPDH (5174, Cell Signaling) and β-actin Rabbit antibodies (ab8227, Abcam). Secondary antibodies used included the following: Alexa Fluor 488 Anti-Mouse; Alexa Fluor 555 Anti-Rabbit; Alexa Fluor 555 Anti-Mouse; and Alexa Fluor 555 Anti-Rabbit (Thermo Fisher Scientific). Additionally, CCK8 (ab228551, Abcam), Dulbecco’s modified Eagle’s medium (DMEM)/F12 culture media, and fetal bovine serum were obtained from Thermo Fisher Scientific.
3
Immunofluorescence analysis of cell-cell junctions
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The following commercially available antibodies were used: claudin-5 (Zymed/Life Technologies), occludin (Transduction Laboratories, Franklin Lakes, NJ, USA), VE-cadherin (Cell Signaling, Danvers, MA, USA), N-cadherin (Transduction Laboratories), β1-integrin (Santa Cruz Biotechnologies, CA, USA), fibronectin (Sigma), calponin (DAKO, Glostrup, Denmark), SMA (Sigma), α-tubulin (Sigma), SRF (Santa Cruz Biotechnologies), phospho-Smad2/Smad3 (Cell Signaling), Smad2/3 (Cell Signaling). Peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Cell Signaling. Cy3-labeled anti-mouse and Cy3-labeled anti-rabbit antibodies were obtained from Jackson ImmunoResearch (Newmarket, UK). SB-431542 was purchased from Sigma. Y-27632 was obtained from Tocris (Bristol, UK). For inhibitor studies control cells were treated with vehicle.
4
Western Blot Analysis of Protein Markers
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Protein samples were isolated from cell lysates (50 μg), and then mixed in reducing buffer, boiled, resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride (PVDF) membrane by electroblotting. The blot was incubated overnight at 4 °C in a blocking solution with primary antibodies directed to the following antigens: GFAP, peroxisome proliferator-activated receptor γ (PPARγ), Wnt1 (Abcam, Cambridge, UK), growth differentiation factor 3 (GDF3; Bioss, Woburn, MA, USA), Smad2/Smad3, phospho-Smad2/Smad3 (Cell Signaling Technology, Danvers, MA, USA), transforming growth factor beta (TGF-β; Abcam, Cambridge, UK), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After the blots were washed with 0.1% Tween® 20 (Sigma-Aldrich) in 1 × PBS, they were incubated with horseradish peroxidase-conjugated secondary antibodies corresponding to each primary antibody, then analyzed by enhanced chemiluminescence detection (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Quantification of protein band intensities in three independent experiments was performed, and the relative ratio under the constant reference β-actin was calculated.
5
Protein Expression Analysis in Cell Lines
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We used primary antibodies targeting DLL3 (1:750; ab103102; Abcam, Cambridge, UK), NOTCH1 intracellular domain (NICD1; 1:500; #3608; Cell Signaling Technology, Danvers, MA, USA), NICD2 (1:5000; #5732; Cell Signaling Technology), NICD3 (1:1000; 55114‐1‐AP; Proteintech, Rosemont, IL, USA), NICD4 (1:1500; #2423; Cell Signaling Technology), E‐cadherin (1:200; sc‐8426; Santa Cruz Biotechnology, Dallas, TX, USA), Vimentin (VIM; 1:200; V6630; Sigma‐Aldrich, St. Louis, MO, USA), Snail (1:1000; #3879; Cell Signaling Technology), phospho‐Smad2/Smad3 (1:1000; #8828; Cell Signaling Technology), Smad2/Smad3 (1:1000; #8685; Cell Signaling Technology), Smad4 (1:1000; #38454; Cell Signaling Technology) and ASCL1 (1:250; #556604; BD Pharmingen, Franklin Lakes, NJ, USA). All analyses included staining with Ponceau S, which confirmed equivalent protein loading. Standardization was performed by measuring actin with the anti–actin antibody (1:1500; A2066; Sigma‐Aldrich).
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