To investigate the underlying mechanism of the scaffolds for osteogenic differentiation of MC3T3-E1 cells, after 7 days of culture, cells were lysed with ice-cold RIPA (Thermo Fisher Scientific) and the total proteins were extracted. BCA protein assay kit (Beyotime, Shanghai, China) was used to measure the concentration of total protein. Protein samples (20 μg) were subjected to electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels and electrotransferred onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA) at 4 °C for 2 h. Then the membranes were blotted using 5% Carnation nonfat dry milk and incubated with anti-collagen I antibody, anti-osteopontin antibody, anti-Wnt-5a antibody, anti-β-catenin antibody and anti-β-actin antibody (all purchased from Abcam, Inc., Cambridge, MA) overnight at 4 °C, respectively. The membranes were washed with phosphate-buffered saline containing 0.1% (v/v) Tween-20 and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. After extensive washing, immunoreactive proteins were visualized using the enhanced chemiluminescence reagent (Thermo Fisher Scientific, Rockford, IL, USA) and were quantitatively analyzed with Quantity-One analysis software (Bio-Rad, Munich, Germany).
Anti wnt5a antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
The Anti-Wnt5A antibody is a laboratory research tool designed to detect the Wnt5A protein. Wnt5A is a secreted signaling protein that plays a role in various cellular processes. The antibody can be used to identify and quantify the expression of Wnt5A in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β catenin antibody, by Abcam (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Anti osteopontin antibody, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Quantity one analysis software, by Bio-Rad (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Anti collagen 1 antibody, by Abcam (1 mentions)
Isoproterenol hydrochloride, by Merck Group (1 mentions)
Anti jnk antibody, by Cell Signaling Technology (1 mentions)
Anti il 1 antibody, by Abcam (1 mentions)
Anti il 18 antibody, by Abcam (1 mentions)
Glutathione peroxidase gsh px detection kits, by Nanjing Jiancheng (1 mentions)
Anti nlrp3 antibody, by Cell Signaling Technology (1 mentions)
Anti il 6 antibody, by Abcam (1 mentions)
Superoxide dismutase sod, by Nanjing Jiancheng (1 mentions)
Anti cyclin d1 antibody, by Abcam (1 mentions)
Anti e2f1 antibody, by Abcam (1 mentions)
6 protocols using anti wnt5a antibody
1
Osteogenic Differentiation Mechanisms in MC3T3-E1 Cells
2
Molecular Mechanisms of SFRP5 in Inflammation
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Recombinant human SFRP-5 was obtained from R&D Systems (MN, USA). Isoproterenol hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Wnt5A antibody, anti-SFRP5 antibody, anti-IL-1 antibody, anti-IL-6 antibody, and anti-IL-18 antibody were purchased from Abcam (Cambridge, UK). An anti-glyceraldehyde-3 phosphate dehydrogenase (GAPDH) antibody was purchased from Bioworld (MN, USA). Anti-NLRP3 antibody and anti-JNK antibody were purchased from Cell Signaling Technology (Danfoss, MA, USA). Superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) detection kits were purchased from Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China).
3
Signaling Pathways in Neuronal Cells
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Amyloid β peptide (1–42; Human Aβ42; Abcam plc., #ab120301), Hoechst 33258 (Sigma-Aldrich, #B2883), recombinant mouse Wnt5a protein (Bio-Techne China Co. Ltd. R&D Systems #645-WN), Roscovitine (ROS; Selleck.cn, #S1153), KN62 (Selleck.cn, #S7422), Calmodulin Kinase IINtide, Myristoylated (Ntide; Merck Millipore, #208921) and Z-VAD-FMK (Selleck.cn, #S7023) were added to the media at the indicated concentrations and time points. For transient gene transfection, DIC5 cortical neurons were transfected using the calcium phosphate transfection method described previously (Kingston et al., 2003 (link)). Lipofectamine 3000 (Thermo Fisher Scientific Inc.) was used for transient gene or siRNA transfection of HEK293 cells.
The following primary antibodies were used: Anti-Wnt7a antibody (Abcam plc., #ab100792), Anti-Wnt5a antibody (Abcam plc., #ab72583), Anti-beta Tubulin antibody (Abcam plc., #ab6046), Anti-Cyclin D1 antibody (Abcam plc., #ab16663), Anti-E2F1 antibody (Abcam plc., #ab94888), phospho-Rb (Ser795) antibody (Cell Signaling Technology, Inc., #9301), Anti-β-Catenin antibody (Abcam plc., #ab32572), Anti-Histone H1 antibody (Abcam plc., #ab71594), Anti-phospho-CaMKII alpha (Thr286)/beta (Thr287; Abcam plc., #ab32678), Flag (Sigma-Aldrich, #F1804).
The following primary antibodies were used: Anti-Wnt7a antibody (Abcam plc., #ab100792), Anti-Wnt5a antibody (Abcam plc., #ab72583), Anti-beta Tubulin antibody (Abcam plc., #ab6046), Anti-Cyclin D1 antibody (Abcam plc., #ab16663), Anti-E2F1 antibody (Abcam plc., #ab94888), phospho-Rb (Ser795) antibody (Cell Signaling Technology, Inc., #9301), Anti-β-Catenin antibody (Abcam plc., #ab32572), Anti-Histone H1 antibody (Abcam plc., #ab71594), Anti-phospho-CaMKII alpha (Thr286)/beta (Thr287; Abcam plc., #ab32678), Flag (Sigma-Aldrich, #F1804).
4
Immunoblotting of WNT signaling proteins
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The total protein from cells was isolated with RIPA lysis and added with PMSF. The protein was electro-blotted onto PVDF membranes for 2 hours at 300 mA. The membrane was inoculated with primary antibody (1:1000) and incubated overnight at 4 °C. After that, the membrane was inoculated with the secondary antibody at RT (room temperature) for 1 hour. Primary antibody anti-WNT3A (1:1000, Abcam, USA) and anti-WNT5A antibody (1:1000, Abcam, USA) are used to detect certain protein. The protein bands were exposed.
5
Protein Expression Analysis in Treated DPCs
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Treated DPCs were dissolved in PRO-PREP™ protein extraction solution (iNtRON Biotech, Seongnam, Korea). Total cell lysates (30 µg) were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) using Towbin buffer. Membranes were then blocked with 5% milk in TBS + 0.1% Tween-20 (Sigma Aldrich, Dorset, UK) and incubated with anti-11β-HSD1 antibody (1:500 dilution, Abcam), anti-vascular endothelial growth factor (VEGF) antibody (1:500 dilution, Abcam), anti-Wnt5a antibody (1:500 dilution, Abcam), or anti-alkaline phosphatase (ALP) antibody (1:500 dilution, Abcam) as primary antibodies and monoclonal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:1000, Beyotime Institute of Biotechnology, Nanjing, China) as a control. Blots were reacted with Immobilon Western reagent (Millipore) and detected using an Amersham Hyperfilm electrochemiluminescence (ECL) assay (GE Healthcare, Buckinghamshire, UK). Signals were detected using the ECL Plus Western blotting detection system (Amersham, Buckinghamshire, UK).
6
Western Blot Protein Analysis Protocol
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Proteins were extracted from cells using cell lysis buffer (Cell Signaling Technology, Danvers, Massachusetts, USA) supplemented with proteinase (Roche, Upper Bavaria, Germany) and phosSTOP phosphatase inhibitor cocktail (Roche). Proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were then probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody, and visualized by ECL (GE Healthcare, Buckinghamshire, UK). Antibodies against phospho-ERK, phospho--Akt, total Akt, phosphor-CRAF, total CRAF, phospho-IGF1R, total IGF1R, phospho-MEK, total MEK, phospho EGFR, Ras, phospho Axl, total B-catenin were purchased from Cell Signaling Technology. Total EGFR and total ERK antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and total Axl was obtained from R&D Systems (Minneapolis, Minnesota, USA). Antibody specific for p-EGFR (1173) was obtained from Novus Biologicals, and antibodies specific for Beta-Tubulin was from Milipore. Anti-Wnt5A antibody was purchased from Abcam (Cambridge, Massachusetts, USA).
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