RIPA lysis buffer (Beyotime) was used for protein extraction. Then protein was treated according to the previous description [12 ]. The primary antibodies were anti-matrix metalloproteinase 2 (MMP2; ab97779; Abcam, Cambridge, MA, USA), anti-matrix metalloproteinase 9 (MMP9; ab76003; Abcam) and anti-FZD5 (ab75234; Abcam). Anti-GAPDH (ab9485; Abcam) was used as a loading control.
Ab75234
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab75234 is a laboratory equipment product. It is designed to perform a specific core function in a research or analytical setting. No further details about its intended use or capabilities are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab9485, by Abcam (2 mentions)
Ab32572, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab110073, by Abcam (2 mentions)
Ab109094, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab97779, by Abcam (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ab11350, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Immobilon ecl substrate kit, by Merck Group (1 mentions)
Ab32072, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Dnmt3a, by Cell Signaling Technology (1 mentions)
Ab92379, by Abcam (1 mentions)
Sc 13082, by Santa Cruz Biotechnology (1 mentions)
Ab95281, by Abcam (1 mentions)
8 protocols using ab75234
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Wnt/β-catenin Pathway
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Total protein was extracted, separated, and transferred to polyvinylidene difluoride (PVDF, Millipore, Molsheim, France) membranes, as described previously.15 (link) After being blocked with 5% non-fat milk, the membranes were probed with primary antibodies overnight at 4 °C, followed by the incubation with horseradish peroxidase (HRP)-conjugated IgG (ab6721, Abcam, Cambridge, UK; dilution 1 : 10 000) as the secondary antibody. Immunoreactive bands were detected using the Immobilon ECL Substrate Kit (Millipore) and analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). Primary antibodies were used as follows: anti-FZD5 (ab75234, Abcam; dilution 1 : 800), anti-β-catenin (ab32572, Abcam; dilution 1 : 5000), anti-phosphorylated (p)-β-catenin (ab11350, Abcam; dilution 1 : 1000), anti-c-Myc (ab32072, Abcam; dilution 1 : 1000), anti-Cyclin D1 (ab16663, Abcam; dilution 1 : 100) and anti-GAPDH (ab181602, Abcam; dilution 1 : 10 000).
3
Quantitative Protein Analysis in Cell Lysates
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For the extraction of total protein, cells were lysed in RIPA lysis buffer on ice. A bicinchoninic acid kit (P0012S, Beyotime Biotechnology Co. Ltd., Shanghai, China) was put to use in order to determine the protein concentration. Subsequently, a protein sample of the same amount was separated via SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Consequently, for 2 h, the membranes were blocked in 5 % non-fat milk. Next, they were probed for a period of 1 h at room temperature using an HRP-conjugated secondary antibody (1:2,000, ab205718, Abcam) and the primary antibodies at 4 °C. The enhanced chemiluminescence reagent (34580, Thermo Fisher Scientific) was put to use to visualize the blot bands. Relative protein level, with GAPDH as the loading control, was determined using the Image J software. Here, the primary antibodies used included FZD5 (1:1000, ab75234, Abcam), DNMT3A (1:1000, 3598, Cell Signaling Technology, Beverly, MA, USA) as well as GAPDH (1:3000, ab9485, Abcam).
4
Immunohistochemical Analysis of FZD5 and PCNA
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In order to facilitate heat-mediated antigen retrieval, the prepared mouse lung tissue sections were first deparaffined, then rehydrated, as well as immersed in EDTA solution. After that, the sections were incubated for a period of 20 min with 3 % H2O2 for 20 min as well as blocked for 30 min with normal goat serum. Afterwards, they were incubated for a period of 16 h at 4 °C having the antibodies of FZD5 (1:200, ab75234, Abcam Inc., Cambridge, MA, USA) as well as PCNA (1:1000, 10205-2-AP, Proteintech Group, Inc., Wuhan, Hubei, China). Correspondingly, the sections were incubated for a period of 30 min at room temperature, having goat anti-rabbit IgG (1:2000, ab205718, Abcam). Here, the sections underwent nuclear staining with hematoxylin and DAB color development, followed by dehydration, xylene clarification, as well as sealing for microscopic examination.
5
Western Blot Analysis of Wnt Pathway Proteins
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The protein expression of Ror2 (105 kDa), FZD5 (65 kDa), CaMKII (50 kDa) β-catenin (86 kDa), cyclin D1 (34 kDa), Wnt5a (45 kDa), GAPDH (37 kDa) and β-actin (43 kDa) were analyzed by western blot analysis using anti-Ror2 rabbit monoclonal (ab92379; Abcam, Cambridge, UK), anti-Frizzled 5 rabbit polyclonal (ab75234; Abcam), anti-CaMKII rabbit polyclonal (sc-13082; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-β-catenin rabbit monoclonal (ab32572; Abcam), anti-cyclin D1 rabbit polyclonal (ab95281; Abcam), anti-Wnt5a rabbit polyclonal (2392; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-GAPDH rabbit monoclonal (5174; Cell Signaling Technology, Inc.) and anti-β-actin rabbit monoclonal (4970; Cell Signaling Technology, Inc.) antibodies according to the manufacturer’s instructions.
6
Quantifying Frizzled Receptor Levels in OPCs
7
Immunohistochemical Analysis of Wnt Pathway
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All tissues were embedded in paraffin. At the temperature at 4°C, the paraffin sections were incubated with the primary anti-Wnt5a (Abcam ab110073, Cambridge, UK, final dilution 1:250), anti-Frizzled-2 (Abcam ab109094, Cambridge, UK, final dilution 1:300), and anti-Frizzled-5 (Abcam ab75234, Cambridge, UK, final dilution 1:250) antibody overnight. Then, a biocatalyst streptavidin-horseradish peroxidase-conjugated as secondary antibody (ZSGB-BIO, Beijing, China) were used, at last with hematoxylin counterstaining and 3′-diaminobenzidine (ZSGB-BIO, Beijing, China). In negative control, phosphate buffered saline (PBS) was used to replace the primary antibody.
8
Immunohistochemical Analysis of Wnt Signaling
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The following antibodies were purchased from Abcam (Cambridge, UK): anti-Wnt5a (ab110073; final dilution 1:250), anti-Frizzled-2 (ab109094; final dilution 1:300), and anti-Frizzled-5 (ab75234; final dilution 1:250). Specimen sections were incubated with the primary antibody overnight at 4°C, then incubated with an appropriate biotinylated streptavidin-horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, Beijing, China), followed by hematoxylin counterstaining and finally visualized with 3′-diaminobenzidine (ZSGB-BIO, Beijing, China). PBS replaced the primary antibody in negative control samples. Positive staining was confirmed in all samples based on layer-specific staining within the hair follicle. Stained sections were examined on NanoZoomer Digital Pathology (Hamamatsu, Japan). For quantitative analysis, five randomly selected images of high-power fields (200× magnification) were digitally captured using a DP 70 CCD camera (Olympus Corp., Tokyo, Japan). Immunopositive cells were counted using Image Pro-Plus software (version 6.0; Media Cybernetics, CA, USA).
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