Cells were lysed into 1× SDS loading buffer (50 mM Tris-HCl pH 6.8, 5% β-mercaptoethanol, 2% SDS, 0.01% bromophenol blue, 10% glycerol) followed by sonication (Bioruptor, 2 × 30 s at high setting). Proteins were solved on a 5–15% gradient Tris–glycine SDS–PAGE gel and semi-dry-transferred to nitrocellulose membranes. The following primary antibodies were used at the indicated dilutions: RAP1 (CST, 2399, 1:1,000); RASGRP3 (CST, 3334, 1:1,000), GAPDH (CST, 5174, 1:10,000); AKT (CST, 34685, 1:5,000); p-S473-AKT (CST, 4060, 1:2,000); ETS1 (CST, 14069, 1,000) and ETV2 (Abcam, ab181847, 1:1,000). All antibody information can be found in the Reporting Summary. Horseradish peroxidase (HRP)-conjugated secondary antibodies and the ECL prime western blotting system (GE Healthcare, RPN2232) were then used. Chemiluminescent signals were captured with a digital camera (Kindle Biosciences) and images of protein bands were taken for quantification using ImageJ.
Ab181847
Manufactured by Abcam
Ab181847 is a recombinant monoclonal antibody targeting KLF5. The antibody is produced in HEK293 cells and is supplied as a liquid.
Automatically generated - may contain errors
Lab products found in correlation
Nb110 89062, by Novus Biologicals (2 mentions)
Df6285, by Affinity Biosciences (2 mentions)
Yp1055, by Immunoway (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Digital camera, by Kindle Biosciences (1 mentions)
Ecl prime western blotting system, by GE Healthcare (1 mentions)
Vimentin, by Proteintech (1 mentions)
Universal hood 2 machine, by Bio-Rad (1 mentions)
N cadherin 22018 1 ap, by Proteintech (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Ab76956, by Abcam (1 mentions)
Ab209484, by Abcam (1 mentions)
Ab255809, by Abcam (1 mentions)
Bf8002, by Affinity Biosciences (1 mentions)
Sds polyacrylamide gel, by Beyotime (1 mentions)
Yt1625, by Immunoway (1 mentions)
Af6265, by Affinity Biosciences (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
5 protocols using ab181847
1
Western Blot Analysis of Cell Signaling
2
Western Blot Analysis of Cell Lysates
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A172 and SHG44 cells were lysed with RIPA buffer for half an hour at 4°C. The supernatant was collected and boiled at 95°C for 5–8 min in SDS loading buffer. Then, they were subjected to electrophoresis in 10% SDS-polyacrylamide gels and transferred to the polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in phosphate-buffered saline (PBS) for 1 h before being incubated with the primary antibody at 4°C overnight. The primary antibodies for western blotting used in this study were GAPDH, ETV2 (ab181847, Abcam), N-cadherin (22018-1-AP, proteintech), and vimentin (10366-1-AP, Proteintech). Then the cells were washed three to four times with 0.1% PBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) for 1 h at room temperature. The membranes were washed in 0.1% PBST four times before exposure. Chemiluminescent HRP substrate was purchased from Millipore (Catalog: WBKLS0500). Images were acquired in a Bio-Rad Universal Hood II machine with Image Lab software.
3
Protein Extraction and Western Blotting
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Total cellular protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (Beyotime) (see Table 2 ). Cytoplasmic and nuclear proteins were separately extracted according to the manufacturer's instructions using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Subsequently, the proteins (12 μg) were separated on 10–12% (w/v) SDS-polyacrylamide gel (Beyotime) and transferred onto a PVDF membrane. The membrane was blocked with 5% (w/v) fetal bovine serum (BSA) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies specific for ETV2 (ab181847, 1:1000, Abcam), OSX (ab209484, 1:1000, Abcam), RUNX2 (ab76956, 1:1000, Abcam), OPN (NB110-89062, 1:1000, Novus), Collagen 1 (ab255809, 1:1000, Abcam), HIF-1α (BF8002, 1:1000, Affinity), PHD1 (EGLN2, DF7918, 1:1000, Affinity), PHD2 (DF6285, 1:1000, Affinity), PHD3 (DF12694, 1:1000, Affinity), VEGFA (66828-1-Ig, 1:1000, Proteintech), ERK1/2 (YT1625, 1:1000, ImmunoWay), pERK1/2 (YP1055, 1:1000, ImmunoWay), VE-Cadherin (AF6265, 1:1000, Affinity), AIF (sc-13116, 1:1000, Santa), Mitofilin (sc-390,707, 1:1000, Santa), and β-actin (ZSGB-Bio). The samples were then incubated with a secondary antibody (1:10,000, ZSGB-Bio) for 1 h and visualized using an ECL kit (Millipore).
4
Paraffin-Embedded Tissue Preparation and Immunofluorescence
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The tissue samples were processed as follows: they were initially fixed in 4% paraformaldehyde for 24 h, then embedded in paraffin, and subsequently sectioned into 5-μm thick slices. For hard tissues, extensive decalcification was conducted using 14% EDTA solution (pH 7.4) for four weeks before embedding.
Hematoxylin-Eosin (H&E) and Masson staining were conducted on all serial sections. For immunofluorescence, sections or cells were incubated with primary antibodies, including ETV2 (ab181847, 1:500, Abcam), CD90 (105,201, 1:100, Biolegend), pERK1/2 (YP1055, 1:100, ImmunoWay), HIF-1α (BF8002, AF1009, 1:100, Affinity), PHD2 (DF6285, 1:100, Affinity), CD31 (AF6191, 1:100, Affinity), OPN (NB110-89062, 1:100, Novus), and Collagen I (AF7001, 1:100, Abcam) overnight at 4 °C. Subsequently, secondary antibodies, including FITC-Conjugated Goat anti-mouse IgG (HA1003, 1:200, HUABIO), Alexa Fluor 594 Anti-rabbit IgG (8889, 1:500, Cell Signaling Technology), Alexa Fluor 488 Anti-rabbit IgG (4412, 1:500, Cell Signaling Technology), and FITC-conjugated goat anti-rabbit IgG (SA00003-11, 1:100, Proteintech), were applied for 1 h. Nuclei were labeled with DAPI (Beyotime) for 5 min and images were captured using a fluorescence microscope (Leica).
Hematoxylin-Eosin (H&E) and Masson staining were conducted on all serial sections. For immunofluorescence, sections or cells were incubated with primary antibodies, including ETV2 (ab181847, 1:500, Abcam), CD90 (105,201, 1:100, Biolegend), pERK1/2 (YP1055, 1:100, ImmunoWay), HIF-1α (BF8002, AF1009, 1:100, Affinity), PHD2 (DF6285, 1:100, Affinity), CD31 (AF6191, 1:100, Affinity), OPN (NB110-89062, 1:100, Novus), and Collagen I (AF7001, 1:100, Abcam) overnight at 4 °C. Subsequently, secondary antibodies, including FITC-Conjugated Goat anti-mouse IgG (HA1003, 1:200, HUABIO), Alexa Fluor 594 Anti-rabbit IgG (8889, 1:500, Cell Signaling Technology), Alexa Fluor 488 Anti-rabbit IgG (4412, 1:500, Cell Signaling Technology), and FITC-conjugated goat anti-rabbit IgG (SA00003-11, 1:100, Proteintech), were applied for 1 h. Nuclei were labeled with DAPI (Beyotime) for 5 min and images were captured using a fluorescence microscope (Leica).
5
Immunofluorescence Staining of DPSCs and HUVECs
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Immunofluorescence staining was performed after culturing the infected DPSCs or HUVECs in EGM for 7 days. The cells were treated with trypsin and seeded at a density of 1 × 104 cells/cm2 within a 24-well plate. After 24 h, the cells were fixed with 4% (v/v) paraformaldehyde for 30 min, followed by treatment with 0.25% (w/v) Triton X-100 for 10 min. After blocking with 3% (w/v) bovine serum albumin (BSA) for 60 min, the cells were incubated with primary antibodies against ETV2 (Cat#Ab181847, Abcam) and VE-Cadherin (Cat#Ab33168, Abcam) overnight at 4 °C. Then, the cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime, Shanghai, China) for 60 min at room temperature. Diamidinophenylindole staining was performed for 5 min before examination under fluorescence microscopy.
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