The cells designated for protein extraction from in vitro osteoclastogenesis and osteoblast differentiation assays were directly lysed in the tissue culture plates at different time points using RIPA Cell Lysis Buffer. Western blotting was performed as described previously33 (link). Antibodies used were as follows: Anti-mouse Morc3 (MBL International. Japan); Anti-mouse NFATc1 (BD Biosciences, USA); Anti-mouse V-ATPase d2 subunit (Produced for the Centre for Orthopaedic Research, UWA38 (link)); Anti-mouse DC-STAMP (Merck Millipore, Germany); Anti-rabbit c-FOS (Cell Signaling Technology, USA); Anti-rabbit Phospho-STAT1 (Tyr 701) (Cell Signaling Technology, USA); Anti-rabbit STAT1 (Cell Signaling Technology, USA); Anti-rabbit Rankl (R&D systems, China); Anti-goat OPG (R&D systems, China); Anti-rabbit β-catenin (Cell Signaling Technology, USA) and Anti-mouse β-Actin (JLA-20) (Developmental Studies Hybridoma Bank. USA). Detection was done by respective peroxidase-conjugated antibodies (Sigma-Aldrich, USA) and chemiluminescence reagent (PerkinElmer Life Sciences).
Rabbit anti β catenin
Manufactured by Cell Signaling Technology
Sourced in United States, China
Rabbit anti-β-catenin is an antibody that recognizes the β-catenin protein. β-catenin is a key component of the Wnt signaling pathway and plays a role in cell-cell adhesion.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti vimentin, by Cell Signaling Technology (9 mentions)
Rabbit anti β actin, by Cell Signaling Technology (7 mentions)
Rabbit anti gsk 3β, by Cell Signaling Technology (6 mentions)
Mouse anti β actin, by Merck Group (6 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Mouse anti β actin, by Cell Signaling Technology (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
Rabbit anti akt, by Cell Signaling Technology (3 mentions)
Mouse anti e cadherin, by Cell Signaling Technology (3 mentions)
Rabbit anti cyclin d1, by Cell Signaling Technology (3 mentions)
Rabbit anti e cadherin, by Cell Signaling Technology (3 mentions)
Rabbit anti snail, by Cell Signaling Technology (3 mentions)
Mouse anti e cadherin, by BD (3 mentions)
Rabbit anti histone h3, by Cell Signaling Technology (3 mentions)
Rabbit anti phospho β catenin, by Cell Signaling Technology (3 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (3 mentions)
92 protocols using rabbit anti β catenin
1
Western Blot Analysis of Osteoclast and Osteoblast Markers
2
Ceramide-Induced β-Catenin Localization
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Cultured hDPCs were seeded on 4-well chamber slides at density of 5×103 cells per well (SPL Life Science, Pocheon, Korea). After starvation, hDPCs were treated with C8-ceramide, oleyl, and stearyl ceramides at concentration of 1,000 µM for 24 hours. After aspirating the medium, cells were rinsed with 1X PBS three times for 5 minutes each time. Then hDPCs were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100 for 10 minutes. After washing with 1X PBS, cells were blocked with 1% BSA in 1X PBS for 1 hour at RT on a shaker. They were incubated with anti-rabbit β-catenin (Cell Signaling Technology) primary antibody diluted at 1:100 in blocking buffer and incubated at 4℃ overnight. On the next day, they were incubated with Alexa Fluor 488 labeled goat anti-rabbit secondary antibody (Invitrogen) diluted at 1:200 in PBST at RT for 1 hour in the dark. These stained cells were mounted with mounting medium containing DAPI to counterstain nuclei. Cells were then observed with a fluorescence microscope (Axiovert 200; Zeiss, Oberkochen, Germany).
3
Western Blot Analysis of Protein Expression
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Alterations in the protein expression levels were analyzed by Western blotting as previously described by our group [42 (link)]. In brief, SKBR3 and ZR75 cells (2 × 106 cells) were seeded and treated with DA and BMS-202, individually and in combination for 48 h. Cell lysates were collected, and equal amounts of protein (30 µg) were resolved on 10% SDS PAGE gels and electroblotted onto PVDF membranes, then probed with the following primary antibodies: anti-rabbit Src family (phospho Y418) (Abcam: ab40660), anti-mouse ErbB2 (Abcam: ab16901), anti-rabbit phosphorylated ErbB2 (Abcam: ab53290), anti-mouse E-cadherin (Cell Signaling: 14,472 S), anti-rabbit vimentin (Cell Signaling: 46,173 S), anti-rabbit β-catenin (Cell Signaling: 8480 S), anti-rabbit phosphorylated β-catenin (Cell Signaling: 4176 S), anti-rabbit AKT (Cell Signaling: 9272 S), anti-rabbit phosphorylated AKT (Cell Signaling: 4060 S), anti-rabbit JNK1/2/3 (Abcam: ab179461). Anti-rabbit GAPDH (Cell Signaling: 8480 S) was used to ensure equal loading of protein samples.
Immunoreactivity was analyzed using the ECL Western blotting substrate (Pierce Biotechnology, Rockford, IL, USA), as described by the manufacturer, and blots were imaged using the iBrightTM CL1000 imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Quantification was done using ImageJ software as previously described by our group [43 ].
Immunoreactivity was analyzed using the ECL Western blotting substrate (Pierce Biotechnology, Rockford, IL, USA), as described by the manufacturer, and blots were imaged using the iBrightTM CL1000 imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Quantification was done using ImageJ software as previously described by our group [43 ].
4
TRPV4 Immunohistochemistry in Guinea Pigs
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Guinea pigs were immunized with a peptide corresponding to the mouse TRPV4 C‐terminus (CDGHQQGYAPKWRTDDAPL) coupled with hemocyanin (Sucram). Antibody reactivity and specificity were confirmed by immunoblotting, in which bands of the correct size were observed in protein extracts of TRPV4‐transfected HEK293 cells, but not pcDNA3.1‐transfected cells. Immunohistochemistry was performed as described.8 Animals (n = 3 per group) were perfused transcardially with heparinized PBS followed by 4% paraformaldehyde in phosphate buffer. The maxilla was dissected out and decalcified with 10% EDTA for 7 days. For histology, paraffin sections were cut and stained with hematoxylin‐eosin. For immunohistochemistry, 10‐µm‐thick frozen sections were incubated with anti‐TRPV4 (2 µg/mL), rat anti‐E‐cadherin (1:200; Takara Bio Inc, Shiga, Japan), and rabbit anti‐β‐catenin (1:400; Cell Signaling Technology) antibodies at 4°C overnight. The antibodies were visualized with fluorochrome‐conjugated secondary antibodies (Alexa Fluor® 488 or 594 donkey anti‐guinea pig/rabbit or rat IgG; 1:400; Jackson ImmunoResearch). F‐actin was visualized with Alexa Fluor® 546 phalloidin (Thermo Fisher Scientific). All sections were observed and analyzed with an Axio Imager 2 microscope with ApoTome or an LSM700 confocal microscope (Carl Zeiss).
5
Gastric Cancer Cell Line Characterization
6
Immunostaining of Brain Samples
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Immunostaining of brain sections or dissociated cells was performed as described previously26 (link),35 (link),64 (link). Primary antibodies used were mouse anti-ARID1B (Abcam, ab57461; Abnova, H00057492-M02), rabbit anti-cleaved caspase-3 (Cell Signaling Technology, #9664), mouse anti-BrdU (BD Biosciences, #555627), rabbit anti-p-histone-H3 (Cell Signaling Technology, #9701), rabbit anti-Ki67 (Cell Signaling, #9129), rabbit anti-β-catenin (Cell Signaling, #8480), chicken anti-GFP (Invitrogen, A10262), mouse anti-Parvalbumin (Sigma-Aldrich, MAB1572), rabbit anti-Cux1 (Sigma-Aldrich, SAB2105137), and rabbit anti-Tbr2 (Sigma-Aldrich, AB2283). Appropriate secondary antibodies conjugated with Alexa Fluor dyes (Invitrogen, A32731, A32727, A32723, A32732) were used to detect primary antibodies. DAPI (Sigma-Aldrich, D9542) was used to stain nuclei. No antigen retrieval was performed in this study.
7
Analyzing β-catenin Levels after Retinal Surgery
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Ten days after surgery (n = 3 eyes/group), retinas were harvested and lysed in RIPA buffer, with protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich, Gillingham, UK). Eluted proteins samples (20 μg) were run on 10% (wt/vol.) SDS-PAGE gels. Samples were probed using rabbit anti-β-catenin (1:2000; Cell Signaling Technology, Danvers, MA, USA) and protein levels compared with β-actin (1:10,000, Santa Cruz Biotechnology, Dallas, TX, USA). Antibodies were validated by the manufacturers for western blot analysis (ESM Table 2 ).
8
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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The cultured cells were rinsed with cold PBS before treated with RIPA lysis buffer at 4 °C for 10 min. Then the mixture was heated at 100 °C for 10 min and centrifuged under 4 °C at 14000 g min−1 for 10 min. The supernatant was removed, and the protein concentration was measured with the BCA method. About 20 μg of protein was loaded in each lane, separated by 10% SDS–PAGE and transferred to the PVDF membrane. The membrane was blocked with 5% non-fat milk powder for 1 h at room temperature before overnight incubation with primary antibodies 4 °C, followed by the secondary antibody. The antibodies were mouse anti-β-tubulin (Cell Signaling, Danvers, MA, USA; CAT 6181), rabbit anti-MBD3 (Cell Signaling, CAT 3896), mouse anti-Flag (Sigma, San Francisco, CA, USA; CAT F1804), rabbit anti-MMP2 (ImmunoWay, Plano, TX, USA; CAT YT2798), rabbit anti-MMP9 (ImmunoWay CAT YT1892), rabbit anti-Vimentin (Cell Signaling, CAT 5741), rabbit anti-N-cadherin (Cell Signaling, CAT 13116), rabbit anti-E-cadherin (Cell Signaling, CAT 3195), rabbit anti-β-catenin (Cell Signaling, CAT 8480), rabbit anti-Snail (Cell Signaling, CAT 3879), rabbit anti-α-SMA (Cell Signaling, CAT 14968), rabbit anti-Smad2/3 (Cell Signaling, CAT 8685), rabbit anti-P-Smad2 (Cell Signaling, CAT 3108), rabbit anti-P-Smad3 (Cell Signaling, CAT 9520), rabbit anti-TGF-β (Cell Signaling, CAT 3709).
9
Immunohistochemical Analysis of Human Placenta
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Immunohistochemical staining of human villi or placenta tissue was performed as previously described using rabbit anti-USP2 (dilution 1:200; Abgent, UK), rabbit anti-Cytokeratin 7 (CK7) (dilution 1:200; proteintech), rabbit anti-HLA-G (dilution 1:200; proteintech). Cells were fixed in 4% paraformaldehyde, permeabilized using 1% Triton X-100 for 30 min, and blocked with 5% BSA for one hour at room temperature. Indirect immunofluorescence was performed using primary antibodies, rabbit anti–β-catenin (1:1,000; Cell Signaling), and secondary antibodies, goat anti-rabbit (1:1,000; proteintech). Nuclear were counterstained with 4′,6-diamindino-2-phenylindole (DAPI; Molecular Probes). Images were acquired using an Olympus microscope. The confocal images were acquired by Laser Scanning Biological Microscope (Olympus). Immunohistochemical staining quantifications were assessed using ImageJ software.
10
Cell culture and Western blot analysis
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Cell lines Huh-7 and PLC/PRF/5 were bought from Cell Bank of Chinese Academy of Sciences. Cells were maintained in DMEM media (#12800017; GIBCO) supplemented with 10% fetal bovine serum (#10437-028; GIBCO) and 1.5 g/L of NaHCO3. The protein samples were blotted with following antibodies: rabbit anti-Cyclin D1 (#2978 S; Cell Signaling Technology) 1:1000, mouse anti-CDK6 (#3136 S; Cell Signaling Technology) 1:1000, rabbit anti-Phospho-Akt(Thr308) (#9275 S; Cell Signaling Technology) 1:600, rabbit anti-Phospho-Akt(Ser473) (#9271 S; Cell Signaling Technology) 1:600, rabbit anti-c-Jun (60A8) (#9165 S; Cell Signaling Technology) 1:1000, rabbit anti-B-Raf (#sc-166; Santa Cruz Biotechnology) 1;400, rabbit anti-Cleaved Notch1 (Val1744) (#4147 S; Cell Signaling technology)1:300, rabbit anti-β-Catenin (#8480 S; Cell Signaling technology) 1:1000, rabbit anti-LC3B (#ab48394; abcam) 1:1000, mouse anti-GSTP1 (#3369 S; Cell Signaling technology) 1:1000, mouse anti-Actin (#HC201; TransGen Biotech) 1:3000. BALB/c nude mice were housed in laminar flow cabinets with free access to food and water in Laboratory Animal Center of Fujian Medical University. All of animal experiments were performed in accordance with the animal protocols and regulations approved by FJMU Experimental Animal Ethics Committee of Fujian Medical University.
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