GFP+ DFSCs or Wnt5a+/GFP+ DFSCs were plated at 1 × 106 cells per well in six-well plates. Total proteins were extracted using RIPA buffer as per the manufacturer’s protocol. Primary antibodies included anti-Wnt5a (1:500; Abcam), anti-Runt-Related Transcription Factor 2 (Runx2, 1:500; Santa Cruz, Dallas, TX, USA), anti-osteocalcin (Ocn, 1:500; Santa Cruz, NM, Dallas, TX, USA) and anti-alkaline phosphatase antibody (ALP, 1:500; Abcam), with anti-glyceraldehyde-3-phosphate dehydrogenase (1:3,000; Abcam) as control. All assays were performed in triplicate.
Anti wnt5a
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-Wnt5a is an antibody that binds to the Wnt5a protein. Wnt5a is a signaling protein involved in various cellular processes. This antibody can be used for the detection and analysis of Wnt5a in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti β catenin, by Abcam (5 mentions)
Anti runx2, by Abcam (3 mentions)
Anti gapdh, by Abcam (3 mentions)
Anti cd9, by System Biosciences (3 mentions)
Anti cd63, by System Biosciences (3 mentions)
Anti cd81, by System Biosciences (3 mentions)
Anti col1a1, by Abcam (2 mentions)
Anti wnt3a, by Abcam (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti apc, by Abcam (2 mentions)
Anti ror2, by Abcam (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase, by Abcam (1 mentions)
Anti osteocalcin ocn, by Santa Cruz Biotechnology (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Anti cox 2, by Santa Cruz Biotechnology (1 mentions)
Anti jnk1, by Santa Cruz Biotechnology (1 mentions)
Anti osx, by Abcam (1 mentions)
Anti col2a1, by Abcam (1 mentions)
Ass3403, by Abcepta (1 mentions)
18 protocols using anti wnt5a
1
Osteogenic Differentiation of Dental Fibroblasts
2
Immunostaining for Liver Protein Localization
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Immunostaining was performed on 3 µm sections after deparaffinization. Microwave or high pressure antigen retrieval was performed in citrate buffer pH 6.0 for 10 min prior to peroxidase quenching with 3% H2O2 in phosphate-buffered saline (PBS) for 10 min. The sections were then washed in water and preblocked with normal goat or rabbit serum for 10 min. Then, slides were incubated, respectively, with anti-Wnt5a (Abcam, USA), anti-JNK1 (Santa Cruz Biotechnology, USA), anti-NF-κB p65 (Santa Cruz Biotechnology, USA), and anti-COX-2 (Santa Cruz Biotechnology, USA) (final concentrations 1∶100, 1∶100, 1∶200, 1∶320, respectively) for over-night at 4°C. The sections were then incubated with biotinylated secondary antibodies (1∶400) for 20 min. Following a washing step with PBS, the avidin–biotin complex was applied. Finally, the sections were rinsed in PBS, developed with diaminoben-zidine tetrahydrochloride substrate for 3 min and counterstained with hematoxylin. Five fields with a final magnification of ×400 were randomly selected for each rat liver, and the Integral Optical Density(IOD) were analyzed by Image Pro Plus 6.0 software.
3
Western Blot Analysis of Tissue-Specific Markers
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After collected cell extract, the protein samples were separated in SDS-PAGE running buffer, and transferred onto polyvinylidene difluoride membrane. After isolation for 1
the corresponding primary antibody was incubated at 4 °C overnight: Anti-Mkx (1/200, Abcam), Anti-Tnmd (1/200, Abcam), Anti-Col1a1 (1/1000, Abcam), Anti-Osx (1/1000, Abcam), Anti-Col2a1 (1/2000, Abcam), Anti-Runx2 (1/1000, Abcam), Anti-Wnt3a (1/1000, Abcam), Anti-Wnt5a (1/1000, Abcam) and Anti-β-catenin (1/10,000, Abcam), and Anti-GAPDH antibody (1/20,000, Abcam), then incubated with horseradish peroxidase (HRP)-conjugated antibodies (Abgent, ASS3403, 1/20,000). The signal was revealed with an ECL-Detection Kit (Millipore, USA).
the corresponding primary antibody was incubated at 4 °C overnight: Anti-Mkx (1/200, Abcam), Anti-Tnmd (1/200, Abcam), Anti-Col1a1 (1/1000, Abcam), Anti-Osx (1/1000, Abcam), Anti-Col2a1 (1/2000, Abcam), Anti-Runx2 (1/1000, Abcam), Anti-Wnt3a (1/1000, Abcam), Anti-Wnt5a (1/1000, Abcam) and Anti-β-catenin (1/10,000, Abcam), and Anti-GAPDH antibody (1/20,000, Abcam), then incubated with horseradish peroxidase (HRP)-conjugated antibodies (Abgent, ASS3403, 1/20,000). The signal was revealed with an ECL-Detection Kit (Millipore, USA).
4
Western Blotting Protocol for Protein Analysis
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The protocol and procedure for western blotting were as described in our previous reports 30 (link), 44 (link), 45 (link). The anti-p-YAP (Ser127), anti-YAP, anti-RalA and anti-β-actin primary antibodies were obtained from Cell Signalling Technology (Danvers, MA, USA). The anti-Wnt5a, anti-Wnt5b, anti-Aggrecan and anti-SOX9 primary antibodies were obtained from Abcam (Cambridge, MA, USA). The anti-CD63, anti-CD9, anti-CD81 and anti-Alix were obtained from System Biosciences (Palo Alto, CA, USA). Based on previous research 46 (link), 47 (link), collagen type II was analysed using 5% (wt/vol) SDS-PAGE with anti-collagen type II primary antibodies (Chemicon®, Merck-Millipore).
5
Western Blot Analysis of Cell Signaling Proteins
6
Wnt Signaling Pathway Regulation Assay
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SR141716 (Rimonabant) and AVE1625 were kindly donated by Sanofi-Aventis (Montpellier, France). It was dissolved in DMSO and added to cells cultures at the indicated concentrations. Anti-β-Catenin, anti-Dvl3, anti-Fzd7, anti-APC, anti-Wnt5A, anti-ROR2, anti-phospho-CaMKII anti LRP5 and anti-Histone H3 were from Abcam. Anti-Cyclin D1 and anti-Lamin A/C were purchased from Becton Dickinson and Sigma-Aldrich, respectively. Anti-acetyl-Histone H4 and anti-acetyl-Histone H3 were from Santa Cruz Biotechnology and Merck Millipore, respectively. Anti-Annexin V FITC conjugated was purchased from Miltenyi Biotec. Primary antibodies not previously reported, secondary HRP-linked goat anti-mouse or goat anti-rabbit IgG, were all from Cell Signalling Technology. All the cell culture reagents were from Sigma–Aldrich, Inc.
7
Protein Expression Analysis in Ocular Surface Cells
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Proteins were extracted from the OS tissues or cultured OS cells by RIPA lysis buffer (Abcam) with protease inhibitor supplement. The concentration of protein in each sample was quantified by BCA assay with a standard assay kit (Thermo Fisher Scientific). Equal amount of protein was loaded for SDS‐PAGE. Proteins in the gels were then transferred to Nitrocellulose membranes (Millipore). Following that, 5% skimmed milk was used as blocking buffer to block the membranes for 30 min at room temperature and then primary antibodies were added for incubation at 4°C overnight. The membranes were washed with TBST three times and then incubated with specific species secondary antibodies (KPL) at room temperature for 2 hours. The membranes were washed again before visualization using an ECL kit. The primary antibodies used for the study were as follows: anti–GLUT1 (1:1000; Santa Cruz); anti–HK2 (1:500; Cell Signaling); (1:800; Cell Signaling); anti–LDHA (1:800; Cell Signaling); anti–Wnt5a (1:800; Abcam); anti–Ror2 (1:800; Abcam); and anti–β‐actin (1:2000; Abcam).
8
Quantifying Protein Expression in Cells
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Western blots were carried out as described previously.22 The following primary antibodies were used: anti‐GAPDH, anti‐PTEN, anti‐WNT5A, and anti‐N‐cadherin (all from Abcam, Cambridge, UK; ab128915, ab32199, ab72583, and ab76011, respectively). IRDye 800CW goat anti‐rabbit IgG and IRDye 680CW goat anti‐mouse IgG (both from LI‐COR Biosciences, Lincoln, NE, USA) were used as secondary antibodies.
9
Protein Expression Analysis in Cells
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Protein lysates of cells were prepared using RIPA buffer supplemented with a proteinase inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA). Protein concentrations were measured using a BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were separated on 4–15% SDS-PAGE gradient gels (Bio-Rad Laboratories, München, Germany) and transferred to PVDF membranes (Merck Chemicals, Darmstadt, Germany). The following first antibodies monoclonal rabbit anti-GAPDH [EPR16891] (Abcam, Cambridge, UK); anti-ATF2 (phosphor T51 + T69) (Abcam, Cambridge, UK), anti-total ATF2 (Abcam, Cambridge, UK); anti-Wnt5A (Abcam, Cambridge, UK) and HSC70 (Santa Cruz, Dallas, TX, USA) were used. Protein expression was analyzed using Image J.
10
Western Blot Analysis of Wnt Pathway
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Cultured cells were lysed in RIPA buffer, and lysates were analyzed using a standard western blot procedure. GAPDH was used as the loading control. PVDF membranes were incubated with anti-GAPDH (1 : 5000), anti-LRP6 (1 : 1000), anti-β-catenin (1 : 1000), anti-Wnt3A (1 : 1000), anti-Wnt7A (1 : 1000), anti-Noggin (1 : 1000), or anti-Wnt5A (1 : 1000) antibodies (Abcam, USA) overnight at 4°C and then washed and incubated for 1 h with secondary antibodies (Abcam, USA).
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