Whole-cell extracts from H460 cells were prepared using cell lysis buffer containing 25 mM HEPES pH 7.5, 0.3 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 20 mM β-glycerophosphate, 0.1 mM Na3VO4, 0.1% Triton X-100 and complete protease inhibitor cocktail (P8340 Sigma, Madrid, Spain). Lysates were centrifuged and the supernatants transferred to a new tube. After adding SDS loading buffer the lysate was boiled for 5 min. 20 µg of protein extract was separated on SDS-polyacrylamide gel and electroblotted to Immobilon P membranes (Millipore, Burlington, VT, USA). Antibodies used were anti-p-ERK5 (3371, Cell Signaling, Danvers, MA, USA), anti-ERK5 (3372, Cell Signaling), anti-p-ERK1/2 (9106, Cell Signaling), anti-fibronectin (sc-71113, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p120 (P17920, BD Biosciences, Franklin Lakes, NJ, USA), γ-catenin (610254, BD Transduction, Franklin Lakes, NJ, USA), anti-β-tubulin (T9026, Sigma, St. Louis, MO, USA), phospho-Smad2/3 (Ser 423/425) and Smad2/3 (FL-425) were from Santa Cruz Biotechnology (Dallas, TX, USA). The signal was detected using enhanced chemiluminiscent (ECL) method (Santa Cruz Biotechnology). After washes the blots were re-incubated with the α-tubulin antibody to normalize for protein load.
γ catenin
Manufactured by BD
Sourced in United States
γ-catenin is a protein that plays a key role in cell-cell adhesion and signaling. It is a member of the catenin family and is involved in the regulation of the Wnt signaling pathway. This protein helps maintain the structural integrity of cells and is essential for various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
β catenin, by BD (4 mentions)
E cadherin, by BD (3 mentions)
β catenin ctd, by BD (2 mentions)
β catenin ntd, by Abcam (2 mentions)
Actin, by Merck Group (2 mentions)
N cadherin, by BD (2 mentions)
Vimentin, by BD (2 mentions)
Anti erk5, by Cell Signaling Technology (1 mentions)
Smad2 3 fl 425, by Santa Cruz Biotechnology (1 mentions)
Anti perk5, by Cell Signaling Technology (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
P8340, by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Protease inhibitor and phosphatase inhibitor tablets, by Roche (1 mentions)
Donkey anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Donkey anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Nupage tris acetate precast polyacrylamide gels, by Thermo Fisher Scientific (1 mentions)
α catenin, by BD (1 mentions)
10 protocols using γ catenin
1
Western Blot Analysis of Signaling Proteins
2
Western Blot Analysis of Cell Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor tablets (Roche, IN, USA). Cell lysates were cleared by centrifugation and protein concentration determined by Bio-Rad Protein Assay kit (Bio-Rad Laboratories, CA, USA). 5–30 µg (as indicated in the figure legends) of total protein in SDS sample buffer was loaded per lane and separated on NuPAGE Tris-Acetate precast polyacrylamide gels (Life Technologies). Detection of proteins was done with the following primary Abs and dilutions: β-catenin CTD (BD Transduction Laboratories, #610153, 1∶2000), γ-catenin (BD Transduction Laboratories, #610253, 1∶5000) β-catenin NTD (Abcam, ab32572, 1∶5000), E-cadherin (BD Transduction Laboratories, #610181, 1∶4000), α-Catenin (BD Transduction Laboratories, #610193, 1∶500), p120-catenin (BD Transduction Laboratories, #610133, 1∶2000), Actin (Sigma-Aldrich, A2066, 1∶2000). Secondary Abs were: donkey anti-mouse IgG-HRP (Santa Cruz Biotechnology, CA, USA, sc-2314, 1∶5000), donkey anti-rabbit IgG-HRP (Santa Cruz biotechnology, sc-2313, 1∶5000). Signals were developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) and quantification of signal intensities was done using the Image Studio Lite software (LI-COR, NB, USA).
3
Immunostaining of Cell Cytoskeleton Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunostaining cells were fixed (4% paraformaldehyde) for 10 minutes at room temperature (RT) and blocked (5% bovine serum albumin (BSA), 5% goat serum, 0.1% Tween-20 in phosphate buffered saline (PBS)) for 1 hour at RT followed by incubation with primary antibody (Ab) (0.5% BSA, 0.5% goat serum, 0.1% Tween-20 in PBS) at 4°C over night. After washing in PBS cells were incubated with fluorescently labeled secondary Ab (0.5% BSA, 0.5% goat serum, 0.1% Tween-20 in PBS) for 30 minutes at RT. For nuclear counterstaining the cells were incubated with DAPI (1 µg/ml) for 10 minutes at RT. Cells stained on cover slips were mounted on slides using Fluorescence Mounting Medium (DAKO, Denmark). The following primary Abs and dilutions were used: β-catenin CTD (BD Transduction Laboratories, BD Biosciences, CA, USA, #610153, 1∶2000), γ-catenin (BD Transduction Laboratories, #610253, 1∶2000) β-catenin NTD (Abcam, Cambridge, UK, ab32572, 1∶250). Secondary Abs used were: Alexa Fluor 488 Donkey Anti-Mouse, (Life Technologies, A-21202, 1∶500), Alexa Fluor 488 Donkey Anti-Rabbit (Life Technologies, A-21206, 1∶500). Images were visualized under a fluorescence microscope (Zeiss Axiovert 200 M, Carl Zeiss, Germany) and acquired with CCD camera (Zeiss Axiocam HR).
4
Immunofluorescence Analysis of Cell-Cell Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monoclonal antibodies against Snail-1, ERK1/2, phospho-ERK1/2, phospho-SMAD1-5-8, and Cav1 were from Cell Signaling Technology; monoclonal antibodies against E-cadherin, β-catenin, and γ-catenin were from BD (Becton-Dickinson Laboratories, Mountain View, CA); monoclonal antibodies against occludin was from Invitrogen (Carlsbad, CA); monoclonal antibodies against tubulin, α-SMA, α-catenin, vimentin, pan-cytokeratin, and fibronectin were from Sigma (Saint Louis, MO); monoclonal anti-N-cadherin and polyclonal anti-ZO-1 were from Zymed (Invitrogen, Carlsbad, CA); polyclonal anti-actin, -SMAD2-3, and -phospho-MEK were from Santa Cruz Biotechnology (CA); and polyclonal anti-phospho SMAD2-3 was from Biosource (Camarillo, CA). Monoclonal anti-CD54 was from Biolegend (San Diego, CA); polyclonal anti-Fsp1 was from Dako (Glostrup, Denmark); monoclonal anti-CD31 from Serotec (Oxford, UK); and polyclonal anti-Ki67 from Abcam (Cambridge, UK). Monoclonal anti-JAM-A clone BV-11 was a gift from Dr. E. Dejana (Milan Italy). Fluor 647?phalloidin and Hoechst 33342 were from Invitrogen (Carlsbad, CA). CI 1040 was from Selleck (Houston, TX).
5
Westerns Blotting for EMT Markers
6
Co-culture of Mesothelial and Ovarian Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mesothelial cells (derived from pleural lavage) were seeded on glass coverslips and allowed to grow until they reached complete confluence. 1.5 × 105 EOC cells detached with 5 mM EDTA, labeled with vital green fluorescent lipophilic tracer DiO (Invitrogen) and washed with PBS were then plated on mesothelial layer in serum free medium for 24 hours.
For immunofluorescence analyses cells plated on coverslips were fixed in PBS-4% paraformaldehyde (PFA) at room temperature (RT), blocked in PBS-1% bovine serum albumin (BSA) and stained as indicated with primary antibodies: anti-α-tubulin-fluorescein isothiocyanate (FITC) (Sigma), phospho-Histone H3 (S10) (Upstate), ZO-1 (Cell Signalling) and γ-catenin (BD Biosciences). Then samples were washed in PBS and incubated with secondary antibodies (Alexa-Fluor 488-, 633- or 546-conjugated anti-mouse or anti-rabbit antibodies; Invitrogen) for 1 hour at RT. PI (5 μg/ml + RNaseA) or TO-PRO-3 iodide (Invitrogen) were used to visualize nuclei and Alexa-Fluor 647- or 546-Phalloidin (Invitrogen) for F-actin staining. Coverslips were mounted with glycerol/0.25% DABCO and analyzed using a the Leica Time Lapse AF6000LX workstation, the TCS-SP2 or the TCS-SP8 Confocal Systems (Leica Microsystems Heidelberg GmbH) interfaced with the Leica Confocal Software (LCS) or the Leica Application Suite (LAS) software.
For immunofluorescence analyses cells plated on coverslips were fixed in PBS-4% paraformaldehyde (PFA) at room temperature (RT), blocked in PBS-1% bovine serum albumin (BSA) and stained as indicated with primary antibodies: anti-α-tubulin-fluorescein isothiocyanate (FITC) (Sigma), phospho-Histone H3 (S10) (Upstate), ZO-1 (Cell Signalling) and γ-catenin (BD Biosciences). Then samples were washed in PBS and incubated with secondary antibodies (Alexa-Fluor 488-, 633- or 546-conjugated anti-mouse or anti-rabbit antibodies; Invitrogen) for 1 hour at RT. PI (5 μg/ml + RNaseA) or TO-PRO-3 iodide (Invitrogen) were used to visualize nuclei and Alexa-Fluor 647- or 546-Phalloidin (Invitrogen) for F-actin staining. Coverslips were mounted with glycerol/0.25% DABCO and analyzed using a the Leica Time Lapse AF6000LX workstation, the TCS-SP2 or the TCS-SP8 Confocal Systems (Leica Microsystems Heidelberg GmbH) interfaced with the Leica Confocal Software (LCS) or the Leica Application Suite (LAS) software.
7
Immunofluorescence Staining of SOX9-Regulated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (A549-pSin-Vector, A549-SOX9, NCI-H460-pSin-Vector, NCI-H460-SOX9, A549-pSuper-Vector, A549-SOX9 sh1#, NCI-H460-pSuper-Vector and NCI-H460-SOX9 sh1#) for immunofluorescence staining were grown and treated in chamber slides, fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized for 10 min with 0.2% Triton X-100 in PBS, and blocked with 2% bovine serum albumin (BSA) for 1 h. Primary antibodies against SOX9 (#ab182579, Abcam), E-cadherin (#610181, BD Biosciences), γ-catenin (#610254, BD Biosciences), N-cadherin (#610920, BD Biosciences), vimentin (#550513, BD Biosciences), and β-catenin (#610154, BD Biosciences) were diluted to 1:400 in PBS containing 1% BSA and incubated for 1 h at room temperature. Secondary antibody was purchased from Life Technologies® (Grand Island, NY), diluted to 1:250 in 1% BSA in PBS and incubated for 1 h. Images were captured with the Nikon® TS2 microscope.
8
Western Blot Analysis of Protein Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell extracts were prepared in a lysis buffer (0.5% Triton X-100, 50 mm β-glycerophosphate, pH 7.2, 0.1 mm sodium vanadate, 2 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 2 μg/ml leupeptin, and 4 μg/ml aprotinin) and the Western blot analysis was carried out as previously described (24 (link)). The following antibodies were used for immunoblotting: γ-catenin (610253, BD Biosciences), HAI-1 (H-1, sc-137159, Santa Cruz Biotechnology), p53 (2524, Cell Signaling Technology), and actin (A3853, Sigma-Aldrich). Aliquots of various protein extracts were resolved on 10% SDS-PAGE gels and transferred to nitrocellulose. The filters were blocked in Tris-buffered saline (10 mm Tris-HCl, pH 7.4, 140 mm NaCl, containing 0.1% Tween-20 (TTBS) and 3% nonfat dry milk and then incubated with the same blocking solution containing the indicated antibodies at 0.5 μg/ml for 16 h. Filters were extensively washed in TTBS, and bound antibodies were visualized with horseradish peroxidase (HRP)-coupled secondary antibodies.
9
Comprehensive Molecular Profiling of MUC1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents were from Sigma-Aldrich unless otherwise noted. The mouse anti-MUC1-ED (catalog number MA1-06503), hamster anti-MUC1-CD (catalog number MA5-11202), rabbit anti-FLAG (catalog number 740001), mouse anti-6XHis (catalog number 37-2900), horseradish peroxidase (HRP)-conjugated rabbit antimouse IgG (catalog number 31450), HRP-conjugated goat-anti-rabbit IgG (catalog number 31460), and HRP-conjugated goat antihamster IgG (catalog number PA1-28823) antibodies were from Thermo Fisher Scientific. The rabbit antibodies to NEU1 (catalog number ab233119), PPCA (catalog number ab217857), and c-Met (catalog number ab216574); and the mouse antibodies to PI3K (p85) (catalog number ab86714), p53 (catalog number ab154036), PDGFRβ (catalog number ab69506), Akt (catalog number ab108202) and phosphorylated Akt (catalog number ab105731) were from Abcam. The mouse antibodies to c-Src (catalog number 612378), EGFR (catalog number 610017), γ-catenin (catalog number 610253), and GST (catalog number 610719) were from BD Biosciences. Glutathione-agarose and Ni-NTA-agarose were from GE Healthcare Bio-Sciences. Biotinylated PNA and HRP-conjugated streptavidin were from Vector Laboratories.
10
Antibody Immunodetection in Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies were used as follows: anti-E-cadherin (1:1000 for western blot and 1:200 for IF; BD Biosciences); anti-α-catenin (1:1000 for western blot and 1:200 for IF; BD Biosciences); β-catenin (1:1000; BD Biosciences); γ-catenin (1:1000; BD Biosciences); α-tubulin (1:4000 for western blot and 1:200 for IF; Cell Signaling); β-actin (1:1000; Cell Signaling); phalloidin (1:200; Invitrogen). All of the secondary antibodies (1:3000 for western blot and 1:300 for IF) were purchased from CWBIO, China. ROCK inhibitor Y27632 was purchased from TOCRIS and used at final concentration of 10 uM. Prolong Gold antifade reagent with DAPI was purchased from Life technologies.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!