For protein analysis, cells were lysed at 4°C for 30 minutes in RIPA buffer (20 nM Tris·HCl, 150 nM NaCl, 1% deoxycholate, 0.1% SDS, 1% Triton X‐100, pH 7.8) supplemented with protease and phosphatase inhibitors cocktail and PMSF (Sigma‐Aldrich). Aliquots of cell lysates containing 20–30 µg of proteins, as evaluated by Bradford, run on 4%–20% Mini‐Protean TGX Stain–Free Gels (BIO‐RAD, Hercules, CA, USA) under reducing conditions and blotted onto PVDF membrane filters using the iBLOT system (Life Technologies). For Western blot analysis, anti‐CD133 (AC133 epitope) (Miltenyi Biotech), anti‐actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐β‐catenin (R&D Systems, Minneapolis, MN, USA), anti‐E‐cadherin (BD Biosciences, Franklin Lakes, NJ, USA) and anti GSK3 alpha and beta (Abcam, Cambridge, UK) Abs were used. Data are expressed as mean ± SD of band intensity normalized to actin of six independent experiments.
Anti β catenin
Manufactured by R&D Systems
Sourced in United States, Morocco
Anti-β-catenin is a laboratory reagent that can be used to detect and quantify the presence of β-catenin, a key signaling protein involved in various cellular processes. It can be utilized in techniques such as Western blotting, immunohistochemistry, and ELISA to identify and analyze the expression levels of β-catenin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
Anti e cadherin, by R&D Systems (1 mentions)
Anti zeb1, by Abcam (1 mentions)
F actin, by Cell Signaling Technology (1 mentions)
Omat x ray film, by Kodak (1 mentions)
Anti snail, by Novus Biologicals (1 mentions)
Anti nephrin, by Santa Cruz Biotechnology (1 mentions)
Anti podocin, by Merck Group (1 mentions)
Anti wnt3a, by Abcam (1 mentions)
Anti p rr, by Abcam (1 mentions)
Foxo1 antibody, by Cell Signaling Technology (1 mentions)
Anti myod, by BD (1 mentions)
Anti myogenin, by BD (1 mentions)
Anti n cadherin, by Merck Group (1 mentions)
6 protocols using anti β catenin
1
Protein Expression Analysis of Stemness Markers
2
Immunofluorescence Staining of Cell Markers
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Cells were fixed and stained according to published methods [17 (link)]. Treated cells were fixed and incubated with the following antibodies: anti-E-cadherin (R&D Systems, 1:500), anti-β-catenin (1:500, R&D Systems), anti-Zeb1 (1:200, Abcam), and F-actin (Cell Signaling). Following primary incubation, conjugation with FITC (Sigma Biochemicals), Cy3 (Sigma Biochemicals), or Alexa Fluor 594 (Invitrogen) secondary antibodies was then performed. The cells were imaged with a Zeiss Axiophot microscope. Images were merged and analyzed using NIH Image J software.
3
Podocyte Protein Expression Analysis by Western Blot
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Western blot analysis was performed as we described previously. In brief, homogenates from cultured podocytes were prepared using sucrose buffer containing protease inhibitor. After boiled for 5 min at 95°C in a 5× loading buffer, 20 µg of total proteins were subjected to SDS-PAGE, transferred onto a PVDF membrane and blocked by solution with dry milk. Then, the membrane was probed with primary antibodies of anti-PRR (1∶1000, Abcam), anti-Wnt3a (1∶1000, Abcam), anti-β-catenin (1∶500, R&D system), anti-snail (1∶500, Novus), anti-nephrin (1∶100, Santa Cruz), anti-podocin (1∶1000, sigma) or anti-β-actin (1∶5000, Santa Cruz) overnight at 4°C followed by incubation with horseradish peroxidase-labeled IgG (1∶5000). The immunoreactive bands were detected by chemiluminescence methods and visualized on Kodak Omat X-ray films. Densitometric analysis of the images obtained from X-ray films was performed using the Image J software (NIH, Bethesda, MD, USA).
4
Western Blot Analysis of Protein Expression
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The protocol of western blot has been described before [33] . Briefly, Aliquots of total lysate (100 µg) in RIPA buffer were run on 8% SDS-PAGE gels before blotted onto PVDF membrane. Then, PVDF membranes were extensively washed with 1X PBS containing 0.5% Tween 20 (PBST) before blocked by blocking solution (5% BSA in PBST) for an hour. Both primary and HRP-conjugated secondary antibodies were diluted 1∶1000 in blocking solution and incubated sequentially with the blot. After extensive washes with PBST, the signals was detected by a chemilminescence kit (Amersham Pharmacia Biotech) and visualized on X-ray films (Super RX, Fuji Medical X-film; Tokyo, Japan). For detection of internal control, all the blots were stripped and washed thoroughly in PBST, then, blocked and incubated with Gapdh or Lamin B1 antibody as described above. The extraction of nuclear protein has been described before [34] (link). Polyclonal and monoclonal FoxO1 antibodies were purchased from Cell Signaling Technology (#9462 and # 2880 respectively). Other antibodies used in this study include anti-β-catenin (MAB1329, R&D systems), anti-Mef2 (SC-133, Santa Cruz), anti-MyoD (554130, BD Pharmingen), anti-Myogenin (556358, BD Pharmingen), Lamin B1 (ab16048, ABcam).
5
Comprehensive Antibody Immunoblotting Protocol
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Following antibodies were used: anti-p21 (1:500), anti-vimentin (1:5,000), anti-E-cadherin (1:250), anti-survivin (1:2,000), anti-phospho-Erk1/2 (1:1,000), and anti-Erk1/2 (1:1,000)were from Cell Signaling Technology (Boston, MA); anti-N-cadherin (1:4,000) was from EMD Millipore (Billerica, MA); anti-β-catenin (1:250) and anti-XIAP (1:1,000) were from R&D Systems (Minneapolis, MN); and anti-β-actin (1:3,000) was from Santa Cruz Biotechnology (Dallas, TX).
6
Immunofluorescent Analysis of Caveolin-1, Galectin-1, and β-Catenin
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PMSCs were
fixed in 10% formalin for 20 min, RT and blocked with 3% BSA for 30
min. Cells were incubated with anti-caveolin-1 (1:200, #7C8, Santa
Cruz Biotechnology), and anti-galectin-1 (1:200, #D608T, Cell Signaling
Technologies) or anti-β-catenin (1:200, #MAB2081, R&D Systems)
overnight at 4 °C. AlexaFlour647-conjugated anti-mouse and AlexaFlour555-conjugated
anti-rabbit secondary antibodies were added at a 1:1000 dilution for
1 h, RT. 4′,6-Diamidino-2-phenylindole (DAPI) was added to
visualize the cell nuclei. Nonspecific mouse and rabbit IgG controls
were performed.
fixed in 10% formalin for 20 min, RT and blocked with 3% BSA for 30
min. Cells were incubated with anti-caveolin-1 (1:200, #7C8, Santa
Cruz Biotechnology), and anti-galectin-1 (1:200, #D608T, Cell Signaling
Technologies) or anti-β-catenin (1:200, #MAB2081, R&D Systems)
overnight at 4 °C. AlexaFlour647-conjugated anti-mouse and AlexaFlour555-conjugated
anti-rabbit secondary antibodies were added at a 1:1000 dilution for
1 h, RT. 4′,6-Diamidino-2-phenylindole (DAPI) was added to
visualize the cell nuclei. Nonspecific mouse and rabbit IgG controls
were performed.
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