Cells were lysed with M-Per Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Equal amounts of the extracts were loaded, subjected to 10% SDS-PAGE, transferred onto nitrocellulose membranes, and probed by antibodies against STAT3 (Cell Signaling), E-cadherin (Cell Signaling), CD133 (Abcam), MEF2D (Abcam), ROCK1 (Abcam), WNT7A (Abcam), KPNA4 (Abcam), VEGF-A (Santa Cruz), Snail (Santa Cruz), Survivin (Santa Cruz) and GAPDH (Santa Cruz) at 4 °C overnight. After incubation with the corresponding secondary antibodies, signals were detected with an ECL detection kit (Amersham Pharmacia Biotech, UK).
Wnt7a
Manufactured by Abcam
Sourced in United States
Wnt7a is a secreted signaling protein that belongs to the Wnt family. It plays a role in cellular processes such as cell fate determination, cell migration, and patterning during embryonic development.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ecl detection kit, by Cytiva (1 mentions)
Mef2d, by Abcam (1 mentions)
Rock1, by Abcam (1 mentions)
Cd133, by Abcam (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Survivin, by Santa Cruz Biotechnology (1 mentions)
Vegfa, by Santa Cruz Biotechnology (1 mentions)
Snail, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Gsk3b, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
10 protocols using wnt7a
1
Protein Expression Analysis in Cells
2
Wnt7a Expression Regulation in Colorectal Cancer
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Total protein was extracted and lysed with RIPA buffer containing protease inhibitor cocktail (Roche). The proteins were separated by SDS-PAGE electrophoresis and transferred to membrane. Membranes were probed with specific primary antibodies against β-actin (Santa Cruz), Wnt7a (abcam), and detected by horseradish peroxidase-conjugated secondary antibodies.
The expression levels of Wnt7a in colorectal cancer cell lines HT-29 and HCT 116 were detected by qRT-PCR.3. The down-regulated Wnt7a expression vector was constructed, and the down-regulated Wnt7a expression cell line was established. The regeneration ability of cancer cells was detected by stem cell ball formation assay, and the influence of plate cloning assay on the proliferation ability of colorectal cancer cells was detected.
The expression levels of Wnt7a in colorectal cancer cell lines HT-29 and HCT 116 were detected by qRT-PCR.3. The down-regulated Wnt7a expression vector was constructed, and the down-regulated Wnt7a expression cell line was established. The regeneration ability of cancer cells was detected by stem cell ball formation assay, and the influence of plate cloning assay on the proliferation ability of colorectal cancer cells was detected.
3
Western Blot Analysis of Corneal Proteins
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Total protein from corneas was extracted by using RIPA lysis buffer. The BCA protein assay kit (Beyotime Institute of Biotechnology) was used for determination of protein content. The extracted proteins (10-20 µg per lane) were separated using 12.5% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (PVDF; MilliporeSigma). The membrane was blocked with 5% bovine serum albumin (BSA) (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature and incubated overnight at 4˚C with primary antibodies against β-catenin (1:500; cat. no. 32572; Abcam), Wnt7a (dilution 1:1,000; cat. no. 183653; Abcam), cyclin D1 (dilution 1:200; cat. no. 16663; Abcam), p-Gsk3b (dilution 1:1,000; 9392, Cell Signaling Technology, Danvers, USA), Gsk3b (dilution 1:1,000; 12456, Cell Signaling Technology, Danvers, USA) and β-actin (dilution 1:1,000; cat. no. 8226; Abcam). On the second day, the membrane was incubated with a goat anti-rabbit IgG H&L HRP-coupled secondary antibody (dilution 1:2,000; cat. no. 6721; Abcam) for 1 h at room temperature. Finally, the bands were visualized by ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc.) via enzyme-linked chemiluminescence using the Omni-ECL Femto Light Chemiluminescence kit (Shanghai Epizyme Biomedical Technology Co., Ltd.) and quantified using ImageJ (version 1.52; National Institutes of Health).
4
Hippocampal Protein Expression Analysis
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Hippocampal tissues were homogenized and dissolved in the ice lysis-buffer containing a cocktail of protein phosphatase and protease inhibitors (21μg/ml aprotinin, 0.5μg/ml leupetin, 4.9mM MgCl2, 1mM sodium-Meta-vanandante, 1% Triton X-100 and 1mM PMSF) to avoid dephosphorylation and degradation of proteins. The samples were centrifuged at 14000rpm at 4°C for 7 mins. The total protein of supernatant was quantified using the Bicinchoninic acid protein assay (Beyotime Biotechnology). 30μg of proteins were resolved using 12% SDS-PAGE, transferred onto PVDF membrane (Merck Millipore). The blot was probed with primary antibodies of NR2B, Arc/Arg3.1, Wnt7a and GAPDH (Abcam, Inc.). Proteins were incubated with secondary antiboby and visualized using the electrochemiluminescence method. All the results were normalized against GAPDH. Each target protein was performed 3 times.
5
Immunofluorescence Imaging of Brain Slices
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Brain slices were fixed with methanol at 4 °C for 10 min and blocked with diluted donkey serum (Jackson ImmunoResearch, West Grove, PA) for 60 min at room temperature. Slides were incubated with primary antibodies of CD31 (1:200, R&D system, Minneapolis, MN), occludin (1:200, Life Technologies, Carlsbad, CA), claudin-5 (1:200, Life Technologies), β-catenin (1:100, Abcam), GFAP (1:500, Millipore), NeuN (1:50, Millipore), and Wnt7a (1:100, Abcam) overnight at 4 °C. After rinsing three times with PBS, brain slices were incubated with the fluorescence conjugated second antibodies for 1 h at room temperature. Immunofluorescence photos were collected by a confocal microscope (Leica, Solms, Germany). For claudin-5 and β-catenin quantification, mean integrated density was measured by ImageJ software (National Institutes of Health). For occludin staining, the gap length was presented as percentage (%) of whole tight junction staining26 (link),27 (link). We randomly chose at least four vessels in the peri-focal region per brain section, and total eight sections per animal. Then we measured the length of vessel and gap by ImageJ software (National Institutes of Health). Gap length was presented as percentage (%) of gap length in whole vessel.
6
Western Blot Analysis of WNT7A Expression
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Tissues were lysed with radio-immunoprecipitation assay cell lysis buffer (1 mmol/ L PMSF, Beyotime, Shanghai, China). The protein contents were measured and the protein sample was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to the polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). The specific primary antibodies WNT7A (1:500, Abcam, MA. USA), GAPDH (1:1000, Cell Signaling Technology, MA, USA) were incubated overnight at 4°C, washed with Tris buffered saline, and subjected to 1 h incubation with the corresponding secondary antibody. The blots were measured with enhanced chemiluminescence. GAPDH was set as the endogenous control to ensure equal protein loading. The blot strength was quantified using ImageJ [26 (link)].
7
Hippocampal Protein Changes in Developing Pups
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The hippocampal protein (CA1 area) from pups at the age of 14 and 21 days was obtained from hippocampus by using homogenizer for 1 minute and lysis for 1 hour on ice. The protein concentration was determined by using the Bicinchoninic Acid (BCA) method. The protein from hippocampus culture was directly harvested after lead treatment with or without Wnt7a by SDS-PAGE sample buffer solution. An equal amount of samples was resolved by an 8.5% SDS-PAGE gel. Resolved proteins were transferred to a PVDF membrane. The non-specific sites were blocked with 5% non-fat dry milk, followed by incubation with primary antibodies (β-actin and Wnt7a were purchased from Abcam, β-catenine and phospho-β-catenine were purchased from Cell Signaling Technology, USA). The membranes were washed three times and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody to detect bands by enhanced chemiluminescence (ECL, GE Healthcare). The bands developed on the films were quantified by a densitometer. All results were normalized against β-actin.
8
Dickkopf-1 Mediated Wnt Signaling Regulation
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RIPA lysis buffer, BCA kit, Fetal bovine serum (FBS), α-minimum essential medium (α-MEM), penicillin and streptomycin were purchased from Beyotime (Beyotime, Beijing, China). The recombinant human Dickkopf-related protein 1 (DKK1) was purchased from MCE (Shanghai, China). The alkaline phosphatase activity measurement kit was purchased from Sigma Company (Sigma-Aldrich, USA).
Antibodies to β-catenin, Wnt7a, Wnt3a, Sp7, Runx2 and β-actin and appropriate secondary antibodies were obtained from Abcam (CA, USA). Reverse transcription system and GoTaq ® 2-Step RT-qPCR system were obtained from Promega (Madison, Wisconsin, USA).
Antibodies to β-catenin, Wnt7a, Wnt3a, Sp7, Runx2 and β-actin and appropriate secondary antibodies were obtained from Abcam (CA, USA). Reverse transcription system and GoTaq ® 2-Step RT-qPCR system were obtained from Promega (Madison, Wisconsin, USA).
9
Protein Expression Analysis and Coimmunoprecipitation
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The total amount of protein in the lysates was determined by the BCA protein assay (Amresco,USA). An equivalent of 50 µg protein was resolved by 10% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and electroblotted to Polyvinylidene Fluoride (PVDF) membrane that was blocked with 5% non-fat dry milk in Tris buffer saline and Tween 20 (TBST) (150 mM NaCl, 50 mM Tris, 0.05% Tween 20). Subsequently, the membranes were probed with primary antibodies, TSP1, p38MAPK, TNFα, TNFR1, Wnt7a, frizzled2 (FZD2), CNTF, CNTFR, spinophilin, and β-actin (Abcam Cambridge, MA, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody(Abcam, Cambridge, UK). After extensive washing, the immunoreactive bands were visualized by ECL reagent (Thermo USA) after exposure on Kodak BioMax lm (Kodak). The band intensities were quanti ed using QuantityOne software. The intensities were expressed as fold-change relative to the GAPDH levels.
For coimmunoprecipitation, the lysates of tissues were incubated with antibodies overnight (4°C) and subsequently with protein G-agarose beads (Millipore, USA) for 5 h (4°C). Followed by washes with lysis buffer, the eluent was separated by SDS-PAGE and electroblotted to PVDF membrane using primary and secondary antibodies.
For coimmunoprecipitation, the lysates of tissues were incubated with antibodies overnight (4°C) and subsequently with protein G-agarose beads (Millipore, USA) for 5 h (4°C). Followed by washes with lysis buffer, the eluent was separated by SDS-PAGE and electroblotted to PVDF membrane using primary and secondary antibodies.
10
Western Blot Analysis of Wnt7a
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Total protein was extracted and lysed with RIPA buffer containing protease inhibitor cocktail (Roche). The proteins were separated by SDS-PAGE electrophoresis and transferred to membrane. Membranes were probed with speci c primary antibodies against β-actin (Santa Cruz), Wnt7a (abcam), and detected by horseradish peroxidase-conjugated secondary antibodies.
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